Methods and compositions of novel triazine compounds

ABSTRACT

The present invention relates to methods and compositions comprising compounds that treat pathophysiological conditions arising from inflammatory responses. In particular, the present invention is directed to compounds that inhibit or block glycated protein produced induction of the signaling-associated inflammatory response in endothelial cells. The present invention relates to compounds that inhibit smooth muscle proliferation. In particular, the present invention is directed to compounds that inhibit smooth muscle cell proliferation by modulating HSPGs such as Perlecan. The present invention further relates to the use of compounds to treat vascular occlusive conditions characterized by smooth muscle proliferation such as restenosis and atherosclerosis.

PRIOR RELATED U.S. APPLICATION DATA

This application is a continuation of U.S. patent application Ser. No.10/400,140, filed Mar. 26, 2003, now U.S. Pat. No. 7,132,423, which is acontinuation-in-part of U.S. patent application Ser. No. 10/390,485,filed Mar. 17, 2003, now U.S. Pat. No. 7,173,032, which is acontinuation of U.S. patent application Ser. No. 10/253,388 filed Sep.23, 2002, now abandoned which claims priority to U.S. ProvisionalApplication Ser. No. 60/324,147 filed Sep. 21, 2001, each of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to triazine compounds. More particularly,the invention relates to methods and compositions for making and usingtriazine compounds.

BACKGROUND OF THE INVENTION

Synthesis of novel compounds leads to new possibilities for discovery ofnovel therapeutic interventions. By using structure and activityrelationship investigations, compounds can be tailored so that thecompounds have at least one activity that can be predicted from itsstructure. Using high-throughput assays allows for the rapiddetermination of the activity of the newly synthesized compounds.

Novel compounds for new therapeutic interventions are needed for manyareas of medicine and disease treatment. For example, chronic and acuteinflammatory conditions form the basis for diseases affecting all organsystems including, but not limited to, asthma, acute inflammatorydiseases, vascular inflammatory disease, chronic inflammation,atherosclerosis, angiopathy, myocarditis, nephritis, Crohn's disease,arthritis, type I and II diabetes and associated vascular pathologies.The incidence of these inflammatory conditions is on the rise in thepopulation as a whole, with diabetes alone affecting 16 million people.

While inflammation in and of itself is a normal immune response, chronicinflammation leads to complications and ongoing system damage due to theinteractions of unknown cellular factors. In particular, chronicinflammation can cause endothelial damage resulting in vascularcomplications. Coronary artery, cerbrovascular and peripheral vasculardisease resulting from atherosclerotic and thromboembolicmacroangiopathy are the primary causes of mortality in chronicinflammatory diseases.

Many humans and animals have limited lifespans and lifestyles because ofconditions relating to lifestyle choices, such as diet and exercise, orbecause of genetic predispositions to develop a disease. For example,vascular smooth muscle cell proliferation is a common consequence ofendothelial injury and is believed to be an early pathogenetic event inthe formation of atherosclerotic plaques or complications related tovascular injury or as a result surgical interventions. Abnormal vascularsmooth muscle cell (SMC) proliferation is thought to contribute to thepathogenesis of vascular occlusive lesions, including arteriosclerosis,atherosclerosis, restenosis, and graft atherosclerosis after organtransplantation.

Percutaneous coronary artery intervention (PTCA) procedures are the mostcommon in-patient hospital procedure in the United States. According tothe American Heart Association, about one-third of the patients thatundergo balloon angioplasty have restenosis of the widened segment ofthe vessel within approximately 6 months. It may be necessary to performanother angioplasty or coronary artery bypass surgery on restenosedarteries. A key feature of restenosis is arm injury response thatresults in activation of an inflammatory cascade and remodeling of thecells both inside and outside the carotid artery wall. This includesexcessive growth of connective tissue and smooth muscle into the lumenof the artery known as neointimal hyperplasia. Currently there are noeffective pharmacological treatments available that control thepathogenesis of vascular occlusive lesions, such as, but not limited to,arteriosclerosis, atherosclerosis, restenosis, and graft atherosclerosisafter organ transplantation. Identification of effective therapeuticswith minimal side effects will restore quality of life without requiringadditional surgical procedures such as coronary artery bypass surgery.

Control or modulation of factors produced by the body in response toinjury, surgery, metabolic factors or loss of control of in feedbackmechanisms, leading to too much or too little of a factor has long beenthe goal of administering pharmacological agents. One disease thatrapidly growing in the industrialized countries is the occurrence ofdiabetes and all of its attendant sequellae. One of the factorsimportant in the damage associated with diabetes is the presence ofglycated proteins.

Glycated proteins and advanced glycation end products (AGE) contributeto cellular damage, particularly, diabetic tissue injury, by at least bytwo major mechanisms; modulation of cellular functions throughinteractions with specific cell surface receptors, and alteration of theextracellular matrix leading to the formation of protein cross-links.Studies suggest that glycated protein and AGE interactions with cellsmay promote inflammatory processes and oxidative cellular injury. AGEincreases lipoprotein oxidisability and atherogenicity. Its binding tomatrix proteins induces synthesis of cytokines and activates cellularmessangers. Diseases where glycated protein and AGE accumulation is asuspected etiological factor include vascular complications of diabetes,microangiopathies, renal insufficiency and Alzheimer's disease.

The exact mechanisms by which high plasma glucose, as seen in diabetes,causes microvascular damage are not completely understood. One potentialmechanism by which hyperglycemia can be linked to microangiopathies isthrough the process of non-enzymatic glycation of critical proteins.Non-enzymatic glycation, i.e. the linking of proteins with glucose,leads to the formation of glycated proteins. The first step in thisglycation pathway involves the non-enzymatic condensation of glucosewith free amino groups in the protein, primarily the epsilon-aminogroups of lysine residues, forming the Amadori adducts. These earlyglycation products can undergo further reactions such as rearrangements,dehydration and condensations to form irreversible advanced glycationend products (AGE). These are a highly reactive group of molecules whoseinteraction with specific receptors on the cell-surface which arethought to lead to pathogenic outcomes.

Other major area of disease of where treatments are needed and for whichadequate and effective therapies do not exist are cellular proliferativedisorders, or disorders caused by unwanted or unintended cellulargrowth. As mentioned, smooth muscle cell (SMC) hyperplasia is a majorevent in the development of atherosclerosis and is also responsible forthe significant number of failure rates following vascular proceduressuch as angioplasty, stent implantation and coronary artery bypasssurgery. In the normal vessel, SMC are quiescent, but they proliferatewhen damage to the endothelium occurs. Naturally occurring growthmodulators, many of which are derived from the endothelium, tightlycontrol SMC proliferation in vivo. When the control becomes unregulated,a pathological state is induced in the subject.

Another major area of unwanted cellular growth, that is unchecked by thebody's regulatory systems, is cancer or oncological conditions. Manytherapies have been used and are being used in an effort to restorehealth or at least stop the unwanted cell growth. Many times,therapeutic agents can have an effect individually, but often,therapeutic regimes require combinations of different pharmacologicalagents with treatments such as surgery or radiation.

There is a present need for treatments of chronic or acute diseases,such as atherosclerosis, unwanted cellular growth or cellularproliferation, diabetes, inflammatory conditions and vascular occlusivepathologic conditions, because occurrence is frequent, the currentlyavailable treatments are costly and the conditions are refractory tomany pharmacological therapies. The mechanisms involved in the controlor prevention of such diseases are not clear and there exists a need forpreventive and therapeutic treatments of these and other diseases Thus,what is presently needed are novel compounds that find utility inmethods and compositions for treatment and prevention of chronic andacute diseases.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions comprisingnovel compounds, primarily based on a substituted triazine core.Disclosed herein are methods for making novel compounds, the compounds,compositions comprising the compounds, and methods and compositions forusing the compounds. The compounds and compositions comprising thecompounds have utility in treatment of a variety of diseases.

Compositions in accordance with the present invention comprise triazinecompounds, analogs, derivatives, and mixtures thereof. Such triazinecompounds comprise the following structure, where N^(A), N^(B) and N^(C)are typically used to represent pendant substituted amino groupsattached to 1,3,5-triazine at the 2, 4 and 6 positions:

An example of such triazine compounds includes compounds having thefollowing structure.

In this example, each pendent amino (NRR′) group can represent simply anNH₂ group or a secondary or tertiary amino group, including a cyclicsecondary amide, and a range of other substituents as described herein.Compositions in accordance with the present invention also compriseanalogs of the tris(amino) compounds, that include intermediatecompounds in the synthesis of the tris(amino) triazine compoundsindicated above, for example diamino chlorotriazine compounds, or aminodiclorotriazine compounds shown below, where N^(A) and N^(B) are pendantsubstituted amino groups as described above.

Compositions in accordance with the present invention also compriseanalogs of the tris(amino) triazine compounds indicated above, includingcompounds that are isolated as byproducts in the synthesis of thetris(amino) triazine compounds, such as bis(amino)alkoxy triazinecompounds as shown below, where E=O or S and the like.

The present invention also comprises compositions used in making thenovel compounds and methods of making the novel compounds disclosedherein.

The present invention is directed to methods and compositions comprisingcompounds that have utility in treatment of pathological conditions. Oneaspect of the present invention comprises compounds and compositionscomprising such compounds in methods for treating diseases related tounwanted cellular proliferation. Many vascular diseases, such ascardiovascular diseases, organ transplant sequellae, vascular occlusiveconditions including, but not limited to, neointimal hyperplasia,restenosis, transplant vasculopathy, cardiac allograft vasculopathy,atherosclerosis, and arteriosclerosis. are caused by or have collateraldamage due to unwanted cellular proliferation, such as smooth musclecell (SMC) hyperplasia. At least one activity of one or more of thesecompounds is that the compound has the activity of affecting thesynthesis of proteoglycans including induction and synthesis ofproteoglycans and active fragments of proteoglycans. Methods compriseadministration of compositions comprising compounds that have at leastthe activity of affecting cellular proliferation and affectingproteoglycan synthesis and activity.

The present invention also comprises methods and compositions comprisingcompounds described herein that have an activity associated withmodulation of glycosidase enzymes and thus, affecting the substrates forsuch enzymes. Glycosidase enzymes and their activity with theirsubstrates, such as proteoglycans or glycated proteins, are aspects of avariety of diseases such as vascular conditions, proteoglycan-associateddiseases, kidney disease, autoimmune disease and inflammatory diseases.Compounds described herein that have an activity that affects theconcentrations of substrates of glycosidase enzymes are used in methodsof treatment of such vascular, inflammatory, metastatic and systemicdiseases.

An embodiment of the present invention comprises methods andcompositions comprising compounds of the present invention for thetreatment and prevention of conditions or diseases that have as anaspect of the disease or condition, inflammation. An aspect of thepresent invention is directed to methods and compositions comprisingcompounds that are effective in inhibiting inflammation, particularlyinflammation associated with the accumulation or presence of glycatedproteins or AGE. Methods of treatment comprise administration ofcompositions comprising having compounds having at least the activity ofmodulating inflammatory reactions that are components of biologicalconditions including, but not limited to, vascular complications of typeI and type II diabetic-induced vasculopathies, other vasculopathies,microangiopathies, renal insufficiency, Alzheimer's syndrome, andinflammation-induced diseases such as atherosclerosis. An aspect of thepresent invention comprises methods and compositions for the treatmentof diseases, preconditions or pathologies associated with inflammatorycytokines and other inflammation related molecules.

Another embodiment of the present invention comprises methods andcompositions comprising compounds that have at least the activity ofcausing cellular death or a cessation of cellular activity, referred toherein as cytotoxic activity. This activity can be used in methods forin vitro or in vivo cytotoxicity. For example, compounds having thisactivity can be selectively delivered to an area within a livingorganism to selectively kill cells in that area. Such methods are usingin treating hyperproliferative cells, such as cancers, or other unwantedcellular growth or cellular activities. One aspect of the inventionprovides compositions comprising compounds that nonselectively killcells. Another aspect of the invention provides compounds thatselectively kill cells, for example, cells that have a particularcellular marker or other identifying characteristic such as metabolicrate or uptake of a particular compound.

The present invention also comprises pharmaceutical compositionscomprising the compounds disclosed herein. Routes of administration anddosages of effective amounts of the compounds and pharmaceuticalcompositions are also disclosed. For example, the compounds of thepresent invention can be administered in combination with otherpharmaceutical agents in a variety of protocols for effective treatmentof disease.

In another aspect, the present invention relates to drug delivering oreluting medical devices that contain or are coated with at least onecompound disclosed herein. The medical device suitable for use with thecompounds of the present invention include, but are not limited to,stents and other medical devices that can provide a substrate fordelivery of at least one compound.

Other aspects of the present invention comprise compositions and methodsfor microarray devices. Such microarray devices and methods comprise avariety of microarrays that may be used, for example, to study andmonitor gene expression in response to treatment with the compounds ofthe present invention. The microarrays may comprise nucleic acidsequences, carbohydrates or proteins that are determinative for specificcells, tissues, species, disease states, prognoses, disease progression,or any other combination of molecules that can be used to determine aneffect of one or more of the compounds of the present invention. Otherembodiments of the present invention comprise methods using databasesand computer applications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. ¹H NMR ofN-(3-Chloro-4-methoxy-phenyl)-N′-cyclohexylmethyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 2. ¹H NMR ofN-Cycloheptyl-N′-(1-ethyl-pyrrolidin-2-ylmethyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 3. ¹H NMR ofN-(3-Chloro-4-methoxy-phenyl)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-(1-propyl-butyl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 4. ¹H NMR ofN-(1-Aza-bicyclo[2.2.2]oct-3-yl)-N′-(3-chloro-4-methoxy-phenyl)-N″-)1-ethyl-pyrrolidin-2-ylmethyl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 5. ¹H NMR ofN2-(3-chloro-4-methoxy-phenyl)-N4-cycloheptyl-N6-methyl-N6-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine.

FIG. 6. ¹H NMR ofN-Cycloheptyl-N′-ethyl-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine.

FIG. 7. ¹H NMR ofN-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-6-pyrrolidin-1-yl-[1,3,5]triazine-2,4-diamine.

FIG. 8. ¹H NMR ofN-Cyclohexylmethyl-N′-(1-ethyl-pyrrolidin-2-ylmethyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 9. ¹H NMR of6-Chloro-N-cycloheheptyl-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine.

FIG. 10. ¹H NMR of(3-Chloro-4-methoxy-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine.

FIG. 11. ¹H NMR ofN-(3-Chloro-4-methoxy-phenyl)-N′-isopropyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 12. ¹H NMR ofN2-(3-chloro-4-methoxy-phenyl)-N4-isopropyl-N6-methyl-N6-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine.

FIG. 13. ¹H NMR of5-{4-(3-Chloro-4-methoxy-phenylamino)-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-pentan-1-ol.

FIG. 14. ¹H NMR of5-[4-(3-chloro-4-methoxy-phenylamino)-6-(methyl-piperidin-4-yl-amino)-1,3,5-triazin-2-ylamino]-pentan-1-ol.

FIG. 15. ¹H NMR of6-Chloro-N,N″-bis-(3-chloro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine.

FIG. 16. ¹H NMR ofN,N′-Bis-(3-chloro-4-methoxy-phenyl)-N″-methyl-N″-(4-methyl-cyclohexyl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 17. ¹H NMR ofN,N′-Bis-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-triamine.

FIG. 18. ¹H NMR ofN-Butyl-N′-(3-chloro-4-methoxy-phenyl)-N″-(1-methyl-piperidin-4-yl)-N-propyl-[1,3,5]triazine-2,4,6-triamine.

FIG. 19. ¹H NMR ofN2-Butyl-N4-(3-chloro-4-methoxy-phenyl)-N6-methyl-N6-piperidin-4-yl-N2-propyl-1,3,5-triazine-2,4,6-triamine.

FIG. 20. ¹H NMR of6-Cyclohexylmethoxy-N,N′-bis-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4-diamine.

FIG. 21. ¹H NMR of(4-Chloro-6-cyclohexylmethoxy-[1,3,5]triazin-2-yl)-(3-fluoro-4-methoxy-phenyl)-amine.

FIG. 22. ¹H NMR ofN,N′-Bis-(3-chloro-4-methoxy-phenyl)-6-cyclohexylmethoxy-1,3,5-triazine-2,4-diamine.

FIG. 23. ¹H NMR of(4-Chloro-6-cyclohexylmethoxy-[1,3,5]triazin-2-yl)-(3-chloro-4-methoxy-phenyl)-amine.

FIG. 24. ¹H NMR of6-Cyclohexylmethoxy-N-(1-ethyl-pyrrolidin-2-ylmethyl)-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine.

FIG. 25. ¹H NMR ofN-(3-Chloro-4-methoxy-phenyl)-6-cyclohexylmethoxy-N′-methyl-N′-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4-diamine.

FIG. 26. ¹H NMR ofN-Azepan-1-yl-N′-(3-chloro-4-methoxy-phenyl)-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 27. ¹H NMR ofN4-(3-chloro-4-methoxy-phenyl)-N6-methyl-N2-perhydro-azepin-1-yl-N6-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine.

FIG. 28. ¹H NMR ofN-Azepan-1-yl-6-chloro-N′-(3-chloro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine.

FIG. 29. ¹H NMR ofN″-(3-chloro-4-methoxy-phenyl)-N,N′-bis-perhydro-azepin-1-yl-1,3,5-triazine-2,4,6-triamine.

FIG. 30. ¹H NMR ofN-(3-Bromo-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 31. ¹H NMR ofN-(1-benzyl-piperidin-4-yl)-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]-2,4,6-triamine.

FIG. 32. ¹H NMR of2-chloro-4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl-amino]-1,3,5-triazin-2-ylamino}-phenol.

FIG. 33. ¹H NMR ofN2-cycloheptyl-N4-((S)-1-ethyl-pyrrolidin-2-ylmethyl)-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine.

FIG. 34. ¹H NMR ofN2-cycloheptyl-N4-((R)-1-ethyl-pyrrolidin-2-ylmethyl)-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine.

FIG. 35. ¹H NMR ofN2-cyclohexylmethyl-N4-((S)-1-ethyl-pyrrolidin-2-ylmethyl)-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine.

FIG. 36. ¹H NMR ofN2-cyclohexylmethyl-N4-((R)-1-ethyl-pyrrolidin-2-ylmethyl)-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine.

FIG. 37. ¹H NMR of({4-cycloheptylamino-6-[((S)-1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile.

FIG. 38. ¹H NMR of({4-cycloheptylamino-6-[((R)-1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile.

FIG. 39. ¹H NMR ofN2-[(1-ethyl-2-pyrrolidinyl]-N4-(3-fluoro-4-methoxyphenyl)-6-[(S)-2-(methoxymethyl)-1-pyrrolidinyl]-1,3,5-triazine-2,4-diamine.

FIG. 40. ¹H NMR of6-Chloro-N-(3-chloro-4-methoxy-phenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine.

FIG. 41. ¹H NMR ofN-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 42. ¹H NMR of4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-1,3,5-triazin-2-ol.

FIG. 43. ¹H NMR ofN2-(3-chloro-4-diethylamino-phenyl)-N4-cycloheptyl-N6-(1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine.

FIG. 44. ¹H NMR ofN2-cycloheptyl-N4-(2-dimethylamino-ethyl)-N6-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine.

FIG. 45. ¹H NMR of({4-cycloheptylamino-6-[1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile.

FIG. 46. ¹H NMR ofN,N′-di-n-propyl-N″-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine.

FIG. 47. ¹H NMR ofN,N′-dicyclopropyl-N″-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine.

FIG. 48. ¹H NMR ofN2-Cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-N6-methyl-N6-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine.

FIG. 49. ¹H NMR ofN2-Cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-N6-methyl-N6-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine.

FIG. 50. ¹H NMR ofN2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-N6-methyl-N6-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine,hydrogen chloride salt.

FIG. 51. ¹H NMR ofN2-(3-chloro-4-diethylamino-phenyl)-N4-cycloheptyl-N6-(1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamineS42-63hydrogen chloride salt.

FIG. 52. ¹H NMR ofN2-cycloheptyl-N4-(1-ethyl-pyrrolidin-2-ylmethyl)-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triaminehydrogen chloride salt.

FIG. 53. Chart showing the effects of compounds in an assay whereglycated human serum albumin (G-HSA) induces IL-6 production.

FIG. 54. Chart showing the effects of compounds in an antiproliferativeassay.

FIG. 55. ¹H NMR of 4-benzyloxy-3-chloro-phenylamine.

FIG. 56. ¹H NMR ofN-(4-benzyloxy-3-chloro-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 57. ¹H NMR ofN-(4-benzyloxy-3-chloro-phenyl)-N′-cycloheptyl-N″-methyl-N″-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 58. ¹H NMR of4-[4-cycloheptylamino-6-(methyl-piperidin-4-yl-amino)-[1,3,5]triazin-2-ylamino]-phenol.

FIG. 59. ¹H NMR of4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5-triazin-2-ylamino}-phenol.

FIG. 60. ¹H NMR of2-Chloro-4-(4,6-dichloro-[1,3,5]triazin-2-ylamino)-phenol.

FIG. 61. ¹H NMR of2-Chloro-4-(4-chloro-6-cycloheptylamino-[1,3,5]triazin-2-ylamino)-phenol.

FIG. 62. ¹H NMR of2-Chloro-4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-phenol.

FIG. 63. ¹H NMR ofN-(1-Benzyl-piperidin-4-yl)-N′-(3-fluoro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-diamine.

FIG. 64. ¹H NMR ofN-(1-Benzyl-piperidin-4-yl)-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-diamine.

FIG. 65. ¹H NMR ofN-Cycloheptyl-N′-(4-methoxy-phenyl)-N″-piperidin-4-yl-[1,3,5]triazine-2,4,6-triamine.

FIG. 66. ¹H NMR of6-Chloro-N-cyclopropyl-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine.

FIG. 67. ¹H NMR ofN-Cyclopropyl-N′-(3-flouro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 68. ¹H NMR ofN-Cyclopropyl-N′-(1-ethyl-pyrrolidin-2-ylmethyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 69. ¹H NMR of6-Chloro-N-(3-chloro-4-methoxy-phenyl)-N′-cyclopropyl-[1,3,5]triazine-2,4-diamine.

FIG. 70. ¹H NMR ofN-Cyclopropyl-N′-(3-chloro-4-methoxy-phenyl)-N″-methyl-″N-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 71. ¹H NMR of6-Chloro-N,N′-bis-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine.

FIG. 72. ¹H NMR ofN-(1-Ethyl-pyrrolidin-2-ylmethyl)-N′,N″-bis-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine.

FIG. 73. ¹H NMR of1-[3-{4-(3-chloro-4-methoxyanilino)-6-cycloheptylamino-1,3,5-triazine-2-yloxy}piperidino]-1-ethanone.

FIG. 74. ¹H NMR ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(2-azolanylmethoxy)-1,3,5-triazine-2,4-diamine.

FIG. 75. ¹H NMR of1-[4-(3-chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazine-2-yl]-piperidin-3-ol.

FIG. 76. ¹H NMR of1-[4-(3-chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazine-2-yl]-piperidin-4-ol.

FIG. 77. ¹H NMR ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(1-methyl-2-azolanylmethoxy)-1,3,5-triazine-2,4-diamine.

FIG. 78. ¹H NMR ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(1-methyl-4-piperidyloxy)-1,3,5-triazine-2,4-diamine.

FIG. 79. ¹H NMR ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(1,4-thiazinan-4-yl)-1,3,5-triazine-4,2-diamine.

FIG. 80. ¹H NMR ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(2-fluorophenoxy)-1,3,5-triazine-4,2-diamine.

FIG. 81. ¹H NMR ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-[2-(2-fluorophenoxy)ethoxy]-1,3,5-triazine-4,2-diamine.

FIG. 82. ¹H NMR ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(6-methyl-2-pyrylmethoxy]-1,3,5-triazine-4,2-diamine.

FIG. 83. ¹H NMR of3-[4-(3-chloro-4-methoxyanilino)-6-cycloheptylamino-1,3,5-triazin-2-yloxy]-2-ethyl-4H-4-pyranone.

FIG. 84. ¹H NMR ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-isopropoxy-1,3,5-triazine-2,4-diamine.

FIG. 85. ¹H NMR of 1-[4-(3-chloro-4-methoxyanilino)-cycloheptylamino-1,3,5-triazin-2-yl]-2-azoloamymethanol.

FIG. 86. ¹H NMR ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(4-methylpiperzino)-1,3,5-triazine-4,2-diamine.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed above and throughout the text areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention.

I. Description of Compounds

In one aspect, the present invention encompasses novel organic compoundsthat are generally described as N², N⁴, N⁶-tris(amino)-1,3,5-triazineswhich are represented by the names in Table 1 and the structuralformulas in the remaining Tables 2 and following. Representativecompounds of this invention can be described by the general structuralformula below, where N^(A), N^(B) and N^(C) are pendant substitutedamino groups attached to 1,3,5-triazines at the 2, 4 and 6 positions.

Thus, the typical compound encompassed by the present invention includestriazine compounds comprising the following structure:

In this typical embodiment, each pendant NR₁R₂, NR₃R₄, and NR₅R₆ aminogroup can represent a primary, secondary, or tertiary amine when bondedto the triazine core, including a cyclic secondary amide substitutent(for example a pyrrolidin-N-yl group), and a range of other substituentsas described herein. Compositions in accordance with the presentinvention also comprise analogs of the tris(amino) compounds, forexample, compounds that are prepared as intermediate compounds in thesynthesis of the tris(amino) triazine compounds indicated above, orcompounds that represent a partially substituted trizaine core. Many ofthe syntheses of triazine compounds of this invention typically usecyanuric chloride C₃N₃Cl₃ as a starting compound, therefore intermediatespecies such as bis(amino) chlorotriazine compounds, or aminodiclorotriazine compounds shown below, where N^(A) and N^(B) are pendantsubstituted amino groups as described above, are also encompassed bythis invention.

Compositions in accordance with the present invention also compriseanalogs of the tris(amino) triazine compounds indicated above, includingcompounds that are isolated as byproducts in the synthesis of thetris(amino) triazine compounds, a general formula of which is shownbelow, where E=O or S. An example of such a compound is abis(amino)alkoxy triazine compound.

In general terms, the compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazine compoundsof the following general structure:

or an ene, a diene, a triene, or an yne derivative thereof; a saturatedderivative thereof; a stereoisomer thereof; or a salt thereof;wherein:

R¹ is in each occurrence independently selected from —H; alkyl,cycloalkyl, alkenyl, cycloalkenyl, cycloalkadienyl, alkynyl, aralkyl,aralkenyl, aralkynyl, heteroalkyl, alkoxy, alkylthio, alkyl amino, ordialkyl amino, each of which having up to 12 carbon atoms and includinglinear or branched derivatives thereof, cyclic derivatives thereof,substituted derivatives thereof, heteroatom derivatives thereof, orheterocyclic derivatives thereof; aryl; heteroaryl; aryloxy; arylthio;halogen; or amino;

G is selected from NR¹ or O;

E is selected from CH or N;

z is an integer from 0 to 3;

X¹ is selected from R¹, NR¹ ₃ ⁺, CN, NO₂, CO₂R¹, C(O)NR¹ ₂, CH═CR¹ ₂,C≡CR¹, C(O)R¹, SO₂R¹, SO₂OR¹, or NC(O)R¹, or X¹ and X² together is afused aryl, pyridine, dioxane, pyrrole, pyrrolidine, furan, or thiophenering; with the proviso that the R¹ moiety of the C(O)R¹ substituent inthe X¹ position excludes amino or dialkylamino when X¹ is C(O)R¹;

X² is selected from R¹; CX_(x)H_(3-x), wherein X is a halogen and x isan integer from 0 to 3; OR¹; SR¹; NR¹ ₂; CN; C(O)OR¹; NC(O)R¹;4-morpholinyl; 4-methyl-1-piperazinyl; OR², wherein R² is selected fromCH₂OCH₃, CH₂OCH₂OCH₃, CH₂OCH₂CH₂OCH₃, CH₂SCH₃, or C(O)R¹; SR³, whereinR³ is selected from CH₂OCH₃, CH₂OCH₂CH₂OCH₃, CH₂OCH₂CH(CH₃)₂,CH₂NHC(O)CH₃, or SR¹; OM or SM, wherein M is selected from Li, Na, K,Mg, or Ca;

AY¹ is halogen, or A is selected from NR¹ or O, and

Y¹ is selected from R¹; CR⁴ ₃; NR⁴ ₂; OR⁴; or SR⁴;

wherein n is an integer from 0 to 8, m is an integer from 1 to 8, Z¹ isindependently selected from CR¹ or N, Z² is independently selected fromCR¹ ₂, NR¹, O, or S, with the proviso that two O or S atoms are notlocated adjacent to each other, and the proviso that no more than two Z²moieties are NR¹;

R⁴ is in each occurrence independently selected from linear or branchedalkyl, cycloalkyl, cycloalkenyl, cycloalkadienyl, alkenyl, alkynyl,aralkyl, aralkenyl, aralkynyl, heteroalkyl, alkoxy, alkylthio,alkylamino, or dialkylamino, each of which having up to 10 carbon atoms,—H, aryl, heteroaryl, aryloxy, arylthio, halogen, amino, NR¹₂-substituted derivatives thereof, OR¹-substituted derivatives thereof,SR¹-substituted derivatives thereof, or halogen-substituted derivativesthereof; and

DY² is halogen, or D is selected from NR¹ or O wherein R¹ is defined asabove, and

Y² is selected from R¹,

wherein Z¹ is independently selected from N or CR⁴ and Z² isindependently selected as defined above, with the proviso that two O orS atoms are not located adjacent to each other, and with the provisothat no more than two Z² moieties are NR¹. The compounds of the presentinvention according to this general description do not include thosethat encompass the unique combination of substituents that would providethe following compounds:

-   -   N-Cycloheptyl-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-naphthalen-2-yl-[1,3,5]triazine-2,4,6-triamine;    -   N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine;    -   [4-(4-Benzyl-piperazin-1-yl)-6-morpholin-4-yl-[1,3,5]triazin-2-yl]-(4-methoxy-phenyl)-amine;    -   N-Cycloheptyl-6-morpholin-4-yl-N′-naphthalen-2-yl-[1,3,5]triazine-2,4-diamine;    -   N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-6-morpholin-4-yl-[1,3,5]triazine-2,4-diamine;    -   N-Cycloheptyl-6-morpholin-4-yl-N′-phenyl-[1,3,5]triazine-2,4-diamine;    -   N-Cycloheptyl-N′-(4-methoxy-phenyl)-6-morpholin-4-yl-[1,3,5]triazine-2,4-diamine;    -   N-Benzyl-N′-cycloheptyl-N″-(4-methoxy-phenyl)-N-methyl-[1,3,5]triazine-2,4,6-triamine;    -   N-(2-[1,3]Dioxolan-2-yl-ethyl)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-naphthalen-2-yl-[1,3,5]triazine-2,4,6-triamine;        or    -   N-Cyclopropyl-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-naphthalen-2-yl-[1,3,5]triazine-2,4,6-triamine.

Because this invention encompass compounds that represent saturatedderivatives of the above general structure, and compounds that includevarious states of unsaturation (for example, -ene, -diene, -triene, and-yne derivatives of the above compounds), then the aryl or pyridyl ringshown in the general formula above can be partially or completedhydrogenated in this invention. As a result, the C₅E ring in the abovestructure can represent a cylcohexyl or piperidynl ring that is X¹ andX² substituted. In general terms, X¹ usually, but not always, representan electron withdrawing group such as halide or nitro, while X² usually,but not always, represents an electron donating group such as alkoxideor amino.

As indicated in the general structure above, AY¹ and DY² typicallyrepresent an NR¹ moiety (where R¹ is defined above), in which case thesesubstituents constitute a portion of amino or substituted amino groupand therefore, the compound itself constitutes a triazine. In this case,Y¹ and Y² may be selected from a wide range of substituents, including,but not limited to, cycloalkyl with up to 10 carbon atoms;

wherein n is from 1 or 2;

linear or branched alkyl with up to 10 carbon atoms; CH₂R¹;(CHR¹)_(x)NR¹ ₂ wherein x is 1-6; CH₂R¹; (CHR¹)_(x)OR¹ wherein x is from1 to 6;

wherein x is from 3 to 5;

CH₂CF₃; (CHR¹)_(x)Z¹ wherein x is from 1 to 6 and Z¹ is selected fromNR¹ ₂,

wherein y is from 3 to 5,

In these examples, R¹ is independently selected as defined above. Theseare merely representative examples of the definitions of the Y¹ and Y²substitutents, and are not intended to be exclusive.

It is also noted that AY¹ together, and DY² together, can also representa wide range of chemical moieties bonded to the triazine core such ashalide or secondary amino groups such as

In the latter cases, the amino substituent groups are termed secondaryamino groups, however upon bonding to the triazine core, the aminonitrogens become tertiary amine moieties. Examaples of AY¹ together andDY² together include, but are not limited to: halide;

wherein x is from 3 to 5;

wherein x is from 0 to 6;

wherein Z² is selected from R¹,

C(O)R¹, C(O)OR¹, pyridinyl, aryl,

wherein x is from 0 to 6, and wherein R¹ is independently selected asdefined above. These are also merely representative examples of thedefinitions of these substituents, and are not intended to be exclusive.

Representative compounds in accordance with the present invention arepresented in Table 1. This table is not intended to be exclusive of thecompounds of the present invention, but rather exemplary of the triazinecompounds that are encompassed by this invention.

TABLE 1 COMPOUND NUMBER COMPOUND NAME 1N²-(4-bromo-1-naphthyl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 2N²-(4-chloro-1-naphthyl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 3N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-quinolinyl)-1,3,5-triazine-2,4,6-triamine 4N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(6-quinolinyl)-1,3,5-triazine-2,4,6-triamine 5N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(8-quinolinyl)-1,3,5-triazine-2,4,6-triamine 6N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-[1-(2-naphthyl)ethyl]-1,3,5-triazine-2,4,6-triamine 7N²-cycloheptyl-N⁴-(3,4-dichlorophenyl)-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 8N²-cycloheptyl-N⁴-(3,4-difluorophenyl)-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 9N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-[4-(trifluoromethoxy)phenyl]-1,3,5-triazine-2,4,6-triamine 10N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(4-fluorophenyl)-1,3,5-triazine-2,4,6-triamine 114-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-amino]benzonitrile 12N²-(4-chlorophenyl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 13N²-(4-bromophenyl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 14 Ethyl4-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-amino]benzoate 15N²-(1,1′-biphenyl-4-yl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 16N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-fluorophenyl)-1,3,5-triazine-2,4,6-triamine 17N²-(3-chlorophenyl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 18N²-(3-bromophenyl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 19 Ethyl3-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-amino]benzoate 20N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(2-fluorophenyl)-1,3,5-triazine-2,4,6-triamine 21N²-(2-chlorophenyl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 22N²-(2-bromophenyl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 23N²-(1,3-benzodioxol-5-yl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 24N²-cycloheptyl-N⁴-(2,3-dihydro-1,4-benzodioxin-6-yl)-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 25N²-cycloheptyl-N⁴-[4-(dimethylamino)phenyl]-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 26N²-[3-chloro-4-(diethylamino)phenyl]-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 27N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-[4-(4-morpholinyl)phenyl]-1,3,5-triazine-2,4,6-triamine 28N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-[4-(4-methyl-1-piperazinyl)phenyl]-1,3,5-triazine-2,4,6-triamine 29N-{4-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)- amino]phenyl}acetamide30 N-{3-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)- amino]phenyl}acetamide31 N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 32N²-cycloheptyl-N⁴-(4-ethoxyphenyl)-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 33N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-[4-(methylthio)phenyl]-1,3,5-triazine-2,4,6-triamine 34N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(2-pyridinyl)-1,3,5-triazine-2,4,6-triamine 35N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(2-methylphenyl)-1,3,5-triazine-2,4,6-triamine 36N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(4-phenoxyphenyl)-1,3,5-triazine-2,4,6-triamine 37N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-methylphenyl)-1,3,5-triazine-2,4,6-triamine 38N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(4-methylphenyl)-1,3,5-triazine-2,4,6-triamine 392-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-amino]-4-methyl-3-thiophenecarboxamide 40N²-(4-chlorophenyl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-N²-methyl-1,3,5-triazine-2,4,6-triamine 413-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-(phenyl)amino]propanenitrile 42N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(4-methoxyphenyl)-N⁶-methyl-1,3,5-triazine-2,4,6-triamine 43N²-cycloheptyl-N⁴-(2,4-difluorophenyl)-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-methyl-1,3,5-triazine-2,4,6-triamine 44[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)(phenyl)amino]acetonitrile 45N²-(3-chlorophenyl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-N²-methyl-1,3,5-triazine-2,4,6-triamine 46N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-methyl-N⁶-[2-(trifluoromethyl)phenyl]-1,3,5-triazine-2,4,6-triamine 47N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-methyl-N⁶-[4-(trifluoromethoxy)phenyl]-1,3,5-triazine-2,4,6-triamine 48N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-N⁶-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine 49N-benzoyl-4-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)- amino]benzenesulfonamide50 N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(2-naphthyl)-1,3,5-triazine-2,4,6-triamine 51N²-ethyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 52N²-(tert-butyl)-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 53N²-benzyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 54N²-cyclooctyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 55N²-cyclohexyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 56N²-cyclopentyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 57N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-6-(1-pyrrolidinyl)-1,3,5-triazine-2,4-diamine 58N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-hexahydro-1H-azepin-1-yl-1,3,5-triazine-2,4-diamine 59N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-octahydro-1(2H)-quinolinyl-1,3,5-triazine-2,4-diamine 60N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-(4-methylcyclohexyl)-1,3,5-triazine-2,4,6-triamine 61N²-(1-ethyl-pyrrolidin-2-ylmethyl]-N⁴-(3-fluoro-4-methoxyphenyl)-6-((S)-2-methoxymethyl-pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine 62N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-6-(4-methyl-1-piperazinyl)-1,3,5-triazine-2,4-diamine 636-(4-acetyl-1-piperazinyl)-N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4-diamine 64 Ethyl4-{4-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-N⁶-[(3-fluoro-4-methoxyphenyl)amino]-1,3,5-triazin-2-yl}-1-piperazinecarboxylate 65N²-(cyclohexylmethyl)-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 66N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-(2-furylmethyl)-1,3,5-triazine-2,4,6-triamine 67N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-(2,2,2-trifluoroethyl)-1,3,5-triazine-2,4,6-triamine 68N²-[2-(dimethylamino)ethyl]-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 69N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-{4-[2-oxo-2-(1-pyrrolidinyl)ethyl]-1-piperazinyl}-1,3,5-triazine-2,4-diamine 70 N²,N⁴-bis[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 71N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-[2-(1-piperidinyl)ethyl]-1,3,5-triazine-2,4,6-triamine 72N⁶-[4-(1,3-benzodioxol-5-ylmethyl)-1-piperazinyl]-N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4-diamine 73N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-[4-(2-pyridinyl)-1-piperazinyl]-1,3,5-triazine-2,4-diamine 741-[3-({4-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-6-[(3-fluoro-4-methoxyphenyl)amino]-1,3,5-triazin-2-yl}amino)propyl]-2- pyrrolidinone75 N²-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-[3-(1H-imidazol-1-yl)propyl]-1,3,5-triazine-2,4,6-triamine 76N²-cycloheptyl-N⁴-ethyl-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 77N²-(tert-butyl)-N⁴-cycloheptyl-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 78N²-benzyl-N⁴-cycloheptyl-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 79N²-cycloheptyl-N⁴-cyclooctyl-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 80N²-cycloheptyl-N⁴-cyclohexyl-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 81N²-cycloheptyl-N⁴-cyclopentyl-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 82N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-6-(1-pyrrolidinyl)-1,3,5-triazine-2,4-diamine 83N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-6-hexahydro-1H-azepin-1-yl-1,3,5-triazine-2,4-diamine 84N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-6-octahydro-1(2H)-quinolinyl-1,3,5-triazine-2,4-diamine 85N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-(4-methylcyclohexyl)-1,3,5-triazine-2,4,6-triamine 86N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-6-[(2S)-2-(methoxymethyl)-1-pyrrolidinyl]-1,3,5-triazine-2,4-diamine 87N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-6-(4-methyl-1-piperazinyl)-1,3,5-triazine-2,4-diamine 886-(4-acetyl-1-piperazinyl)-N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4-diamine 89ethyl-4-{4-(cycloheptylamino)-6-[(3-fluoro-4-methoxyphenyl)amino]-1,3,5-triazin-2-yl}-1-piperazinecarboxylate 90N²-cycloheptyl-N⁴-(cyclohexylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 91N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-(2-furanylmethyl)-1,3,5-triazine-2,4,6-triamine 92N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-(2,2,2-trifluoroethyl)-1,3,5-triazine-2,4,6-triamine 93N²-cycloheptyl-N⁴-[2-(dimethylamino)ethyl]-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 94N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-6-{4-[2-oxo-(1-pyrrolidinyl)ethyl]-1-piperazinyl}-1,3,5-triazine-2,4-diamine 95N²-cycloheptyl-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 96N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-[2-(1-piperidinyl)ethyl]-1,3,5-triazine-2,4,6-triamine 976-[4-(1,3-benzodioxol-5-ylmethyl)1-piperazinyl]-N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4-diamine 98N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-6-[4-(2-pyridinyl)-1-piperazinyl]-1,3,5-triazine-2,4-triamine 991-[3-({4-(cycloheptylamino)-6-[(3-fluoro-4-methoxyphenyl)amino]-1,3,5-triazin-2-yl}amino)propyl]-2-pyrrolidinone 100N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-[3-(1H-imidazol-1-yl)propyl]-1,3,5-triazine-2,4,6-triamine 101(3-Chloro-4-methoxy-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine 1026-Chloro-N-(3-chloro-4-methoxy-phenyl)-N′-cyclohexylmethyl-[1,3,5]triazine-2,4-diamine 103N-(3-Chloro-4-methoxy-phenyl)-N′-cyclohexylmethyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine 1046-Chloro-N-(3-chloro-4-methoxy-phenyl)-N′-(1-propyl-butyl)-[1,3,5]triazine-2,4-diamine 105N-(3-Chloro-4-methoxy-phenyl)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-(1-propyl-butyl)-[1,3,5]triazine-2,4,6-triamine 106N-(3-Chloro-4-methoxy-phenyl)-N′-isopropyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine 107N²-(3-chloro-4-methoxy-phenyl)-N⁴-isopropyl-N⁶-methyl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine 1085-{4-(3-Chloro-4-methoxy-phenylamino)-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-pentan-1-ol 1095-[4-(3-chloro-4-methoxy-phenylamino)-6-(methyl-piperidin-4-yl-amino)-1,3,5-triazin-2-ylamino]-pentan-1-ol 110N-Butyl-6-chloro-N′-(3-chloro-4-methoxy-phenyl)-N-propyl-[1,3,5]triazine-2,4-diamine 111N-Butyl-N′-(3-chloro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-N-propyl-[1,3,5]triazine-2,4,6-triamine 112N²-Butyl-N⁴-(3-chloro-4-methoxy-phenyl)-N⁶-methyl-N⁶-piperidin-4-yl-N²-propyl-1,3,5-triazine-2,4,6-triamine 1132,4-Dichloro-6-cyclohexylmethoxy-[1,3,5]triazine 114(4-Chloro-6-cyclohexylmethoxy-[1,3,5]triazin-2-yl)-(3-fluoro-4-methoxy-phenyl)-amine 1156-Cyclohexylmethoxy-N,N′-bis-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4-diamine 1166-Cyclohexylmethoxy-N-(1-ethyl-pyrrolidin-2-ylmethyl)-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine 117(4-Chloro-6-cyclohexylmethoxy-[1,3,5]triazin-2-yl)-(3-chloro-4-methoxy-phenyl)-amine 118N,N′-Bis-(3-chloro-4-methoxy-phenyl)-6-cyclohexylmethoxy-1,3,5-triazine-2,4-diamine 119N-(3-Chloro-4-methoxy-phenyl)-6-cyclohexylmethoxy-N′-methyl-N′-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4-diamine 1206-Chloro-N,N″-bis-(3-chloro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine 121 N,N′-Bis-(3-chloro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine 122N,N′-Bis-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-triamine 123N-(3-Bromo-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine 124(4,6-Dichloro-[1,3,5]triazin-2-yl)-(3-fluoro-4-methoxy-phenyl)-amine 1256-Chloro-N-cyclohexylmethyl-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine 126N-Cyclohexylmethyl-N′-(1-ethyl-pyrrolidin-2-ylmethyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine 1276-Chloro-N-cycloheheptyl-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine 128N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-6-pyrrolidin-1-yl-[1,3,5]triazine-2,4-diamine 129N-Cycloheptyl-N′-ethyl-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine 130N-Cycloheptyl-N′-(1-ethyl-pyrrolidin-2-ylmethyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine 1312-[4-chloro-6-(3-chloro-4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-propane-1,3-diol 1322-{4-(3-chloro-4-methoxy-phenylamino)-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-propane-1,3-diol 1336-Chloro-N-(3-chloro-4-methoxy-phenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine 134N-(1-benzyl-piperidin-4-yl)-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]-2,4,6-triamine 135N²-(3-chloro-4-methoxy-phenyl)-N⁴-cycloheptyl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine 136N²-(3-chloro-4-methoxy-phenyl)-N⁴-cycloheptyl-N⁶-(1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine 137N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine 1382-chloro-4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl-amino]-1,3,5-triazin-2-ylamino}-phenol 139N²-cycloheptyl-N⁴-((S)-1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 140N²-cycloheptyl-N⁴-((R)-1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 141N²-cyclohexylmethyl-N⁴-((S)-1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 142N²-cyclohexylmethyl-N⁴-((R)-1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine (42, Scheme 23)143 ({4-cycloheptylamino-6-[((S)-1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile (43, Scheme 24) 144({4-cycloheptylamino-6-[((R)-1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile 145N²-[(1-ethyl-2-pyrrolidinyl]-N⁴-(3-fluoro-4-methoxyphenyl)-6-[(S)-2-(methoxymethyl)-1-pyrrolidinyl]-1,3,5-triazine-2,4-diamine 146N²-(3-chloro-4-methoxy-phenyl)-N⁴-cycloheptyl-N⁶-methyl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine 1474-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-1,3,5-triazin-2-ol 148N-(1-Aza-bicyclo[2.2.2]oct-3-yl)-N′-(3-chloro-4-methoxy-phenyl)-N″-)1-ethyl-pyrrolidin-2-ylmethyl)-[1,3,5]triazine-2,4,6-triamine 149N²-(3-chloro-4-diethylamino-phenyl)-N⁴-cycloheptyl-N⁶-(1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine 150N²-cycloheptyl-N⁴-(2-dimethylamino-ethyl)-N⁶-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine 151({4-cycloheptylamino-6-[1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile 152N-Azepan-1-yl-6-chloro-N′-(3-chloro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine 153N″-(3-chloro-4-methoxy-phenyl)-N,N′-bis-perhydro-azepin-1-yl-1,3,5-triazine-2,4,6-triamine 154N-Azepan-1-yl-N′-(3-chloro-4-methoxy-phenyl)-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine 155N⁴-(3-chloro-4-methoxy-phenyl)-N⁶-methyl-N²-perhydro-azepin-1-yl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine 156N,N′-di-n-propyl-N″-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine 157N,N′-dicyclopropyl-N″-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine 158N²-Cycloheptyl-N⁴-(3-fluoro-4-methoxy-phenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine 159N²-Cycloheptyl-N⁴-(3-fluoro-4-methoxy-phenyl)-N⁶-methyl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine 160N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine, hydrogen chloridesalt 161 [N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]trizaine-2,4,6-triamine, hydrogen chloridesalt 162 N²-(3-chloro-4-diethylamino-phenyl)-N⁴-cycloheptyl-N⁶-(1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine 163N²-(3-chloro-4-diethylamino-phenyl)-N⁴-cycloheptyl-N⁶-(1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine hydrogen chloridesalt 164N²-cycloheptyl-N⁴-(1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine hydrogen chloride salt 165N²-(cyclohexylmethyl)-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(4-fluoro-3-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine hydrogen chloridesalt 166({4-cycloheptylamino-6-[(1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile hydrogen chloride salt 167N²-cycloheptyl-N⁴-(3-fluoro-4-methoxy-phenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine maleate salt 168N²-cycloheptyl-N⁴-(3-fluoro-4-methoxy-phenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine citrate salt 169N²-cycloheptyl-N⁴-(3-fluoro-4-methoxy-phenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine succinate salt 170N-(3-Bromo-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine hydrogen chloridesalt

In general terms, the compositions in accordance with the presentinvention also comprise tris(amino) triazine compounds of the followingstructure:

wherein R₁ to R₆ represent H, alkyl, aryl, alkenyl, alkynyl, aralkyl,aralkenyl, aralkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroaryl,halide, alkoxy, aryloxy, alkylthio, arylthio, silyl, siloxy, amino,alkylamino, dialkylamino and the like, including straight or branchedchain derivatives thereof, cyclic derivatives thereof, substitutedderivatives thereof, heteroatom derivatives thereof, heterocyclicderivatives thereof, functionalized derivatives thereof, salts thereof,isomers thereof, or combinations thereof.

For example, a typical substituent R₁ to R₆ is a substituted alkyl, inwhich the substituent is a heterocyclic derivative. Examples ofnitrogen-containing heterocyclic moieties include, but are not limitedto groups such as pyridinyl (derived from pyridine, bonded through aring carbon), piperidinyl (derived from piperidine and bonded throughthe ring nitrogen atom or a ring carbon), and pyrrolidinyl (derived frompyrrolidine and bonded through the ring nitrogen atom or a ring carbon).

Examples of substituted or functionalized derivatives of R₁ to R₆include, but are not limited to, moieties containing substituents suchas acyl, formyl, hydroxy, acyl halide, amide, amino, azido, acid,alkoxy, aryloxy, halide, carbonyl, ether, ester, thioether, thioester,nitrile, alkylthio, arylthio, sulfonic acid and salts thereof, thiol,alkenyl, alkynyl, nitro, imine, imide, alkyl, aryl, combinationsthereof, and the like. Moreover, in the case of alkylated derivatives ofthe recited moieties, the alkyl substitutent may be pendant to therecited chemical moiety, or used for bonding to the amine nitrogenthrough the alkyl substituent.

Examples of chemical moieties R₁ to R₆ of the present invention furtherinclude, but are not limited to: H; methyl; ethyl; propyl; butyl;pentyl; hexyl; heptyl; octyl; ethenyl; propenyl; butenyl; ethynyl;propynyl; butynyl; cyclobutyl; cyclopentyl; cyclohexyl; cyclobutenyl;cyclopentenyl; cyclohexenyl; phenyl; tolyl; xylyl; benzyl; naphthyl;pyridinyl; furanyl; tetrahydro-1-napthyl; piperidinyl; indolyl;indolinyl; pyrrolidinyl; 2-(methoxymethyl) pyrrolidinyl; piperazinyl;quinolinyl; quinolyl; alkylated-1,3-dioxolane; triazinyl; morpholinyl;phenyl pyrazolyl; indanyl; indonyl pyrazolyl; thiadiazolyl; rhodaninyl;thiolactonyl; dibenzofuranyl; benzothiazolyl; homopiperidinyl;thiazolyl; quinonuclidinyl; isoxazolidinonyl; any isomers, derivatives,or substituted analogs thereof; or any substituted or unsubstitutedchemical groups such as alcohol, ether, thiol, thioether, tertiaryamine, secondary amine, primary amine, ester, thioester, carboxylicacid, diol, diester, acrylic acid, acrylic ester, methionine ethylester, benzyl-1-cysteine ethyl ester, imine, aldehyde, ketone, amide, ordiene.

Further examples of chemical moieties R₁ to R₆ of this inventioninclude, but are not limited to, the following species or substituted oralkylated derivatives of the following species, covalently bonded to theamine nitrogen: furan; tetrahydrofuran; indole; piperazine; pyrrolidine;pyrrolidinone; pyridine; quinoline; anthracene; tetrahydroquinoline;naphthalene; pyrazole; imidazole; thiophene; pyrrolidine; morpholine;and the like. One feature of the recited species or substituted oralkylated derivatives of these species, is that they may be covalentlybonded to the amine nitrogen in any fashion, including through thependant substituent or alkyl group, through the heteroatom asappropriate, or through a ring atom as appropriate, as understood by oneof ordinary skill in the art.

The chemical moieties R₁ to R₆ of the present invention also include,but are not limited to, cyclic alkanes and alkenes and include bridgedand non-bridged rings. Examples of bridged rings include, but are notlimited to, groups such as norbornyl; norbonadienyl, adamantyl;6-azabicyclo[3.2.1]octanyl; 3-azabicyclo[2.2.2]octanyl, and the like.

In one embodiment of the present invention, NR₁R₂, NR₃R₄, or NR₅R₆ arederived from a cyclic secondary amine. Examples of a cyclic aminochemical moiety of the present invention include, but are not limited topiperidine; 4-benzyl-piperidine; 3-piperidinemethanol; moropholine;4-piperidinopiperidine; 1-(2-amino-methyl)-piperazine;decahydroquinoline; 1,2,3,4-tetrahydro-pyridoindole (either aminemoiety); 3-amino-5-phenyl pyrazole; 3-aminopyrazole; histidinol;hexamethyleimine; 4-hydroxypiperidine; 2-piperidinemethanol; 1, 3,3-trimethyl-6-azabicyclo[3.2.1]octane; 3-pyrrolidinol;1-methylpiperazine; 2-ethyl-piperidine; 1,2,3,4-tetrahydroisoquinoline;3-aminopyrrolidine; 2, 6-dimethylmorpholine; 2, 3,4-tetrahydroisoquinoline; 1, 2, 3, 4-tetrahydroquinoline;1-(2-methoxyphenyl) piperazine; 2,6-dimethylpiperazine (either aminemoiety); iminodibenzyl; 5-methoxytryptamine; 4,4′-bipiperidine;1-(2-hydroxyethyl) piperazine; 4-methylpiperidine; and the like.

Importantly, the general structure of the present invention encompassesall states of saturation of the substitutents shown, such as all ene,diene, triene, and yne derivatives of any substitutent. The generalstructure also encompasses all conformational isomers, regioisomers, andstereoisomers that may arise from a particular set of substitutents. Thegeneral structure also encompasses all enantiomers, diastereomers, andother optical isomers whether in enantiomeric or racemic forms, ormixtures of stereoisomers.

Preparation of the Focused Library of Compounds

Many of the compounds of this invention were prepared in a parallelsynthetic procedure according to the methods described below. Examplesof compounds prepared by the parallel synthesis techniques are providedin Table 2. These preparations involve reacting the individual aminecompounds (monomers) with cyanuric chloride, which are also presented inTable 2, along with the chemical structures of compounds prepared by theparallel synthesis methods.

A library of compounds was synthesized according to the presentinvention to afford substituted N², N⁴, N⁶-tris(amino)-1,3,5-triazines,as follows. The design of the compound library was based primarily onstructure 95 shown below. That is, the design of the N², N⁴,N⁶-tris(amino)triazines was focused so that only one of the pendantamino groups (N^(A), N^(B), or N^(C) in the structure above) was changedduring each synthesis, while the other two groups were held constant.The combination of the specific amines employed produced a library ofcompounds of novel composition. Initially, the library was developedusing methyl-(1-methyl-piperidin-4-yl)-amine, holding the cycloheptyland m-fluoroanisidyl groups constant (in structure 95 below). Thesynthesis of the triazines around methyl-(1-methyl-piperidin-4-yl)-aminewas not optimized, and the amine was subsequently replaced with(1-ethyl-pyrrolidin-2-yl)-methylamine which provided a more tractablesynthesis.

The library of N²,N⁴,N⁶-tris(amino)-1,3,5-triazines was prepared basedon the strategy of changing only one pendant amino group per synthesis,and based on the parent structure 95 shown above. The library wasdivided into three subgroups: Libraries I, II, and III (shown in Table2). Library I includes compounds having unchanged N^(B) and N^(C) groupsbut different N^(A) groups (6). The pendant amino group N^(A) waschanged according to the specific examples listed below. Library IIincludes compounds having unchanged N^(A) and N^(C) groups and differentN^(B) groups (7). Pendant amino group N^(B) was changed according to thespecific examples listed below. Library III includes compounds havingunchanged N^(A) and N^(B) groups and different N^(C) groups (8). Thependant amino group N^(C) was changed according to the specific exampleslisted below.

The N², N⁴, N⁶-tris(amino)-1,3,5-triazine compound structures that arepresented in Tables 2 and following were generated using ISIS-Draw™version 2.4.0.20, and were generated with the option to displayunspecified hydrogen atoms if not shown, however, not all hydrogen atomswere displayed in the structures shown. In all structures presented inany text, table, scheme or figure herein, any hydrogen atoms that arerequired for any atom to attain its usual valence, whether a carbon atomor a heteroatom, should be inferred if it is not specifically indicatedin a structure.

One method of preparation of the compounds is shown in the scheme below.The compounds were prepared by reacting cyanuric chloride sequentiallywith monomers of primary or secondary amines to afford the desired1,3,5-triazine derivatives [1,2,3,4]. Thus, the amine starting compoundsthat are used to react with cyanuric chloride are termed “monomers.” TheN², N⁴, N⁶-tris(amino-substituted)-1,3,5-triazines were prepared withoutthe need for purification between each step of the synthesis, and thefinal product was isolated by standard procedures. Purification wasaccomplished using flash column chromatography as needed. It is withinthe skill of the art of organic synthesis to prepare, isolate and purifythese organic compounds described herein, and to modify the synthesesshown. For example, it is possible to synthesize the compounds of thepresent invention by using an excess of any monomers of primary orsecondary amines in any of the three steps shown in Scheme 1, such thatthe excess monomer, serves as both substituent for the triazine core, aswell as a base, in which case i-Pr₂NEt base can be excluded.

The pendant amino groups can be substituted by functional groupsdepicted as R₁ to R₆ groups in Scheme 1. The degree of functionality ofa pendant amino group is determined by the structure and complexity ofthe amine monomer, and will affect the overall molecular diversity ofthe N², N⁴, N⁶-tris(amino-substituted)-1,3,5-triazines. A wide range ofamine monomers may be used in this invention. Once bonded to thetriazine core, the pendant amino groups can be described as secondary ortertiary substituted, depending on the degree of substitution at thenitrogen atom.

Table 2 presents charts of N², N⁴, N⁶-tris(amino)-1,3,5-triazinecompounds of Libraries I-III of this invention, respectively, along withthe amine precursor monomers used in the preparation of the compounds.General procedures and synthetic procedures are detailed in Example 1-5.The sequence in which each monomer is added in Scheme 1 is alsopresented in Table 2, where Monomer 1 is added first, Monomer 2 addedsecond, and Monomer 3 is added third. While not intending to be bound bythe following statement, it is believed that this order of addition issignificant, because each synthetic stop necessarily involves thereaction of a monomer with a different triazine precursor. That is,monomer 1 reacts with cyanuric chloride, monomer 2 reacts with an aminodichloro(triazine), and monomer 3 reacts with a diaminochloro(triazine), as shown in Scheme 1. Thus, the order in which themonomers are employed is based on the general synthetic principle thatthe relative nucleophilicity and/or basicity of monomers 1-3 used in thesynthetic scheme should generally increase from monomer 1 to monomer 3.This strategy permits the most nucleophilic and/or basic amine monomerto be reacted with the more sterically congested and presumablyless-reactive diamino chloro(triazine), where its greater reactivity mayassist the reaction proceeding to completion. In some cases, more thanone order of monomer addition will provide the desired product, but thereaction sequences provided in Table 2 represents the optimum syntheticmethods presently known.

Note that only in a general sense do the substituents indicated asN^(A), N^(B), and N^(C) in the general structures above correspond withthe actual N², N⁴, N⁶-nomenclature of the N², N⁴,N⁶-tris(amino)triazines. Because the order in which N², N⁴, and N⁶substituents are assigned a 2-, 4-, or 6-position on the triazine coreis dependent on the name of each amino group in the molecule, it is notalways true that one particular amino group always appears as an N², N⁴,or N⁶ substituent, even when only a single substituent is being permutedat one position. For example, many of the compounds of Table 2 containboth cycloheptyl amino and 3-fluoro-4-methoxyphenyl amino groups, yetthese groups take on different 2-, 4-, or 6-positions as a function ofthe name of the third substituent on the triazine core. As a result, thesyntheses are discussed in terms of permuting amino groups at onependant N^(A), N^(B), or N^(C) position (rather than N², N⁴, or N⁶position) in the structure above, while maintaining the other aminogroups constant. Further, note that the compound names used in theTables, Claims and specification were typically generated usingBeilstein's Autonom™ 4.01.188, as well as the earlier CD “stand-alone”version of Beilstein's Autonom™, Autonom 2000. Typically, the compoundnames generated in this fashion were used, regardless of whether thecompound name is an IUPAC, CAS, Beilstein, or other nomenclature. Ineach case however, the names unambiguously identify the compoundspecified.

A. Amino Groups Derived from Monomer 1

The sequence of monomer reaction with the triazine core, shown in Scheme1, is Monomer 1, Monomer 2, and Monomer 3, added in that order. Thus, anamino dichloro(triazine) is formed from Monomer 1 and cyanuric chloride.For the first pendant amino group derived from Monomer 1 and cyanuricchloride, the Monomer 1 amine used and proposed included primarily, butnot always, aryl amines, specifically phenyl, naphthyl, naphthylalkyl,quinolinyl, heteroaryl derivatives, and the like.

Specific examples of Monomer 1 used to produce the first pendant aminogroup in N², N⁴, N⁶-tris(amino-substituted)-1,3,5-triazines, and their[Chemical Abstract Registry numbers] include, but are not limited to,4-chloroaniline [106-47-9], 3,4-ethylenedioxaniline [22013-33-8],4-bromoanline [106-40-1], ethyl 4-aminobenzoate [94-09-7],4-fluoro-aniline [371-40-4], 4-aminobiphenyl [92-67-1], 3-fluoroaniline[372-19-0], 2-aminonaphthalene [91-59-8], 3-chloroaniline [108-42-9],4-morpholinoaniline [2524-67-6], 3-bromoaniline [591-19-5],4′-aminoacetanilide [122-80-5], ethyl 3-aminobenzoate [582-33-2]m-aminoacetanilide [102-28-3], 2-fluoroaniline [348-54-9], m-anisidine[536-90-3], 2-chloroaniline [[95-51-2], p-phenetidine [156-43-4],2-bromoaniline [615-36-1], 4-(methylthio)aniline [104-96-1],3,4-(methylendioxy)aniline [14268-66-7], 2-aminopyridine [504-29-0],o-toluidine [95-53-4], 2,4-difluoro-N-methylaniline [138564-16-6],4-phenoxyaniline [139-59-3], N-phenylglycinonitrile [3009-97-0],m-toluidine [108-44-1], 3-chloro-N-methylaniline [7006-52-2],p-toluidine 106-49-0], 2-(methylamino)benzotrifluoride,4-chloro-N-methylaniline [932-96-7], 4-aminobenzonitrile [873-74-5],3-anilinopropionitrile, [1075-76-9], tetracaine [94-24-6],N-methyl-p-anisidine [5961-59-1], 3-chloro-p-anisidine [5345-54-0],sulfabenzamide [127-71-9], 3-aminoquinoline [580-17-6],1-amino-4-bromonaphthalene [2298-07-9], 6-aminoquinoline [580-15-4]1-amino-4-chloronaphthalene, [4684-12-2] 8-aminoquinoline [578-66-5],S-(−)-1-(2-naphthyl)-ethylamine [3082-62-0], 3,4-dichloroaniline[95-76-1], 3,4-difluoroaniline [3863-11-4],N-methyl-4-(trifluoromethoxy)aniline [41419-59-4],4-(trifluoromethoxy)aniline [461-82-5],2-amino-4-methylthiophene-3-carboxamide [4651-97-2],N,N-diethyl-N′-phenethylenediamine [1665-59-4],1-(4-amino-phenyl)-4-methylpiperazine hydrochloride [94520-33-9],2-chloro-N′,N′-diethyl-1,4-phenylenediamine monohydrochloride[196938-07-5] 2-(dimethylamino)ethyl 4-aminobenzoate [11012-47-2],N,N-dimethyl-1,4-phenylenediamine [1665-95-4].

B. Amino Groups Derived from Monomer 2

The reaction of Monomer 2 with a preformed amino dichloro(triazine)provides an intermediate diamino chloro(triazine) in the synthesis ofN², N⁴, N⁶-tris(amino-substituted)-1,3,5-triazines. Thus, for bondingthe second pendant amino group to the triazine core, the Monomer 2 amineused and proposed included amines, specifically alkyl (C₁-C₁₂, straightchain or branched), cycloalkyl (C₃-C₁₀ ring size), azacyclo (C₂-C₁₀),and benzyl amine derivatives. The ring of the cycloalkyl andazacycloamine, and phenyl ring of the benzyl derivatives can beoptionally substituted with one or more moieties, or a combination ofmoieties, such as, alkyl, alkenyl, alkynyl, phenyl, benzyl, halo, cyano,nitro, hydroxy, thioxy, alkoxy, aryloxy, haloalkyloxy, alkylthio,arylthio, amino, alkyl amino, aryl amino, acyl, carboxyl, amido,sulfonamido, sulfonyl, sulfate, sulfonic acid, morpholino,thiomorpholino, piperazinyl, pyridyl, thienyl, furanyl, pyrroyl,pyrazoyl, phosphate, phosphonic acid, phosphonate and the like. Thesegroups can be represented in protected or unprotected forms used instandard organic synthesis.

In addition, any monomer described that has a stereocenter includes itsenantiomers, diastereomers, and optical isomers whether in enantiomericor racemic forms, or mixtures of stereoisomers. This is to include allof the 1,3,5-triazine derivatives and their stereoisomers presentedherein that are formed as a result of using optically active, scalemicor racemic monomers.

Specific examples of Monomer 2 used to attach the second pendant aminogroup in the synthesis of N², N⁴,N⁶-tris(amino-substituted)-1,3,5-triazines, and their corresponding[Chemical Abstract Registry numbers] include, but are not limited to,ethylamine [75-04-07], cyclohexanemethylamine [3128-02-8]tert-butylamine [75-64-9], furfurylamine [617-89-0], benzylamine[100-46-9], 2,2,2-trifluroethylamine [753-90-2], cyclooctylamine[5452-37-9N,N-dimethylethylenediamine cyclohexylamine [108-91-8],cyclopentylamine [1003-03-8], 1-(2-aminoethyl)-piperidine [26116-12-1],1-acetylpiperazine [13096-96-3], pyrrolidine [123-75-1],1-piperonylpiperazine [32231-06-4], hexamethyleneimine [111-49-9],1-(2-pyridyl)piperazine [34803-66-2], decahydroquinoline (cis/trans)[2051-28-7], 1-methylpiperazine [109-01-3], 1-(3-aminopropyl)-imidazole[5036-48-6], ethyl 1-piperazine carboxylate [120-43-4],4-methylcyclohexylamine (cis/trans) [6321-23-9],1-(3-aminopropyl)-2-pyrrolidine [7663-77-6],2-(aminomethyl)-ethy-lpyrrolidine [26116-12-1], (+)-S-2-(methoxymethyl)pyrrolidine [63126-47-6], 1-(pyrrolidineocarbonylmethyl) piperazine[339890-45-4].

C. Amino Groups Derived from Monomer 3

The reaction of Monomer 3 with a preformed diamino chloro(triazine)provides the final step in the synthesis of N², N⁴,N⁶-tris(amino-substituted)-1,3,5-triazines. Thus, for bonding the thirdpendant amino group to the triazine core, the Monomer 3 used andproposed consisted of amines, specifically alkyl (C₁-C₁₂, straight chainor branched), cycloalkyl (C₃-C₁₀ ring size), azacyclo (C₂-C₁₀), andbenzyl amine derivatives. The ring of these cycloalkyl-, azacycloamine,and phenyl ring of the benzyl derivatives can be optionally substitutedwith one or more moieties, or a combination of moieties such as groupsas alkyl, alkenyl, alkynyl, phenyl, benzyl, halo, cyano, nitro, hydroxy,thioxy, alkoxy, aryloxy, haloalkyloxy, alkylthio, arylthio, amino, alkylamino, aryl amino, acyl, carboxyl, amido, sulfonamido, sulfonyl,sulfate, sulfonic acid, morpholino, thiomorpholino, piperazinyl,pyridyl, thienyl, furanyl, pyrroyl, pyrazoyl, phosphate, phosphonicacid, phosphonate and the like.

In addition, any monomer described that has a stereocenter includes itsenantiomers, diastereomers, and optical isomers whether in enantiomericor racemic forms, or mixtures of stereoisomers. This is to include allof the 1,3,5-triazine derivatives and their stereoisomers presentedherein that are formed as a result of using optically active, scalemicor racemic monomers.

Specific examples of Monomer 3 used to attach the third pendant aminogroup in the synthesis of N², N⁴,N⁶-tris(amino-substituted)-1,3,5-triazines, and their corresponding[Chemical Abstract Registry numbers] used in the synthesis of the N²,N⁴, N⁶-tris(amino-substituted)-1,3,5-triazine derivatives include, butare not limited to, ethylamine [75-04-07], cyclohexanemethylamine[3128-02-08], tert-butylamine [75-64-9], furfurylamine [617-89-0],benzylamine [100-46-9], 2,2,2-trifluroethylamine [753-90-2],cyclooctylamine [5452-37-9], N,N-dimethylethylenediamine,cyclohexylamine [108-91-8], cyclopentylamine [1003-03-8],1-(2-aminoethyl)-piperidine, [26116-12-1], 1-acetylpiperazine[13096-96-3], pyrrolidine [123-75-1], 1-piperonylpiperazine[32231-06-4], hexamethyleneimine [111-49-9], 1-(2-pyridyl)piperazine[34803-66-2], decahydroquinoline (cis/trans) [2051-28-7],1-methylpiperazine [109-01-3], 1-(3-aminopropyl)-imidazole [5036-48-6],ethyl 1-piperazine carboxylate [120-43-4], 4-methylcyclohexylamine(cis/trans) [6321-23-9], 1-(3-aminopropyl)-2-pyrrolidine [7663-77-6],2-(aminomethyl)-ethyl-pyrrolidine [26116-12-1], (+)-S-2-(methoxymethyl)pyrrolidine [63126-47-6], 1-(pyrrolidineocarbonylmethyl) piperazine[339890-45-4].

In addition to the parallel synthetic procedures used to prepare thecompounds of Table 2, Table 1 also provides other exemplary triazinecompounds of the present invention, which were synthesized individuallyrather than using parallel syntheses. The complete preparation andproperties of these compounds are presented in the Examples, wheredetails of the synthetic procedures used are provided. The syntheticprocedures for these compounds involve both the substitution of chloridegroups on cyanuric chloride, as well as various chemical modificationsof these groups once bonded to the trizine core. In particular, thisinvention also encompasses salts of the neutral triazine compounds, asprovided in the Examples and the Tables.

In another aspect of this invention, compounds of the present inventioninclude, but are not limited to, those having the following formula:

or an ene, a diene, a triene, or an yne derivative thereof; a saturatedderivative thereof; a stereoisomer thereof; or a salt thereof;wherein:

R¹ is in each occurrence independently selected from —H; linear orbranched alkyl with up to 10 carbon atoms; or cycloalkyl with up to 10carbon atoms;

X¹ is selected from m-F, m-Cl, m-Br, m-I, m-CN, m-NO₂, M-SO₂R¹, orm-SO₂OR¹, or X¹ and X² together is a fused benzene, pyridine, or dioxanering;

X² is selected from p-OR¹, p-SR¹, p-NR¹ ₂, p-OM, or p-SM, wherein M isselected from Li, Na, K, Mg, or Ca;

Y¹ is selected from cycloalkyl with up to 10 carbon atoms; linear orbranched alkyl with up to 10 carbon atoms; CH₂R², wherein R² is acycloalkyl with up to 10 carbon atoms; or

wherein n is 1 or 2;

AY² is selected from a halogen or OR¹, or

A is NR¹ and Y² is selected from R¹,

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In a further aspect of this invention, compounds of the presentinvention include, but are not limited to, those having the followingformula:

or an ene, a diene, a triene, or an yne derivative thereof, a saturatedderivative thereof; a stereoisomer thereof; or a salt thereof;wherein:

R¹ is in each occurrence independently selected from —H; linear orbranched alkyl with up to 10 carbon atoms; cycloalkyl with up to 10carbon atoms; or aryl;

E is CH or N;

n is an integer from 0 to 3;

X¹ is selected from —H, m-F, m-Cl, m-Br, m-I, m-CN, m-NO₂, m-SO₂R¹, orM-SO₂OR¹, or X¹ and X² together is a fused benzene or pyridine ring;

X² is selected from —H, o-Cl, o-Br, p-OR¹, p-SR¹, p-NR¹ ₂, p-F, p-Cl,p-Br, p-CF₃, p-C(O)OR¹, p-OM, or p-SM, wherein M is selected from Li,Na, K, Mg, or Ca;

A is selected from NR¹ or O, wherein Y¹ is selected from cycloalkyl withup to 10 carbon atoms, linear or branched alkyl with up to 10 carbonatoms, or

when A is NR¹, and wherein Y¹ is selected from R¹ or CH₂R¹ when A is O;or AY¹ is selected from a halogen,

DY² is a halogen, or D is NR¹ and Y² is selected from

or (CHR¹)_(x)NR¹ ₂, wherein x is an integer from 1 to 6.

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In yet another aspect of this invention, compounds of the presentinvention include, but are not limited to, those having the followingformula:

or an ene, a diene, a triene, or an yne derivative thereof; a saturatedderivative thereof; a stereoisomer thereof; or a salt thereof;wherein:

R¹ is in each occurrence independently selected from —H; linear orbranched alkyl with up to 10 carbon atoms; cycloalkyl with up to 10carbon atoms; aryl; or (CH₂)_(x)CN, wherein x is an integer from 0 to 6;

E is CH or N;

n is an integer from 0 to 3;

X¹ is selected from —H, m-F, m-Cl, m-Br, m-I, m-CN, m-NO₂, m-SO₂R¹,M-SO₂OR¹, m-NC(O)R¹, or o-F, or X¹ and X² together is a fused benzene,pyridine, or dioxane ring;

X² is selected from —H, o-Cl, o-Br, o-CF₃, o-R¹, p-OR¹, p-SR¹, p-NR¹ ₂,p-F, p-Cl, p-Br, p-CF₃, p-CN, p-C(O)OR¹, p-NC(O)R¹, p-(4-morpholinyl),or p-(4-methyl-1-piperazinyl);

AY¹ is a halogen, or A is NR¹ or O and Y¹ is selected from cycloalkylwith up to 10 carbon atoms, cycloalkyl with up to 10 carbon atomssubstituted with R¹, linear or branched alkyl with up to 10 carbonatoms, CH₂R¹, (CHR¹)_(y)OR¹, wherein y is an integer from 1 to 6,

or AY¹ together are

wherein x is an integer from 3 to 5; and

DY² is a halogen, or D is NR¹ and Y² is selected from

cycloalkyl with up to 10 carbon atoms, cycloalkyl with up to 10 carbonatoms substituted with R¹, linear or branched alkyl with up to 10 carbonatoms, CH₂R¹,

wherein x is an integer from 3 to 5,

CH₂CF₃, (CHR¹)_(z)Z¹, wherein z is an integer from 1 to 6, and Z¹ isselected from NR¹ ₂,

wherein x is an integer from 3 to 5,

or NY²R¹ together is selected from

wherein Z² is selected from R¹, C(O)R¹, C(O)OR¹, pyridinyl, aryl,

wherein q is an integer from 0 to 6.

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

As an additional aspect of this invention includes compounds of thepresent invention include, but are not limited to, those having thefollowing formula:

or an ene, a diene, a triene, or an yne derivative thereof, a saturatedderivative thereof; a stereoisomer thereof, or a salt thereof;wherein:

R¹ is in each occurrence independently selected from —H; linear orbranched alkyl with up to 10 carbon atoms; or cycloalkyl with up to 10carbon atoms;

X¹ is selected from H, m-F, m-Cl, m-Br, m-I, m-CN, m-NO₂, m-SO₂R¹, orm-SO₂OR¹;

X² is selected from o-R¹, p-OR¹, p-SR¹, p-NR¹ ₂, p-OM, or p-SM, whereinM is selected from Li, Na, K, Mg, or Ca;

Y¹ is selected from cycloalkyl with up to 10 carbon atoms or

and

Y² is selected from linear or branched alkyl with up to 10 carbon atoms,cycloalkyl with up to 10 carbon atoms, or

and R² is —H; or NY²R² together is selected from

wherein x is an integer from 3 to 5,

wherein q is an integer from 0 to 6, or

wherein Z² is selected from R¹ or

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In still another aspect of this invention, compounds of the presentinvention include, but are not limited to, those having the followingstructural formula:

or an ene, a diene, a triene, or an yne derivative thereof; a saturatedderivative thereof; a stereoisomer thereof; or a salt thereof,wherein:

R¹ is in each occurrence independently selected from —H; linear orbranched alkyl with up to 10 carbon atoms; or cycloalkyl with up to 10carbon atoms;

X¹ is in each occurrence independently selected from —H, m-F, m-Cl,m-Br, m-I, m-CN, m-NO₂, M-SO₂R¹, or m-SO₂OR¹;

X² is in each occurrence independently selected from o-CH₃, p-OR¹,p-SR¹, p-NR¹ ₂, or p-OM or p-SM, wherein M is selected from Li, Na, K,Mg, or Ca;

Y¹ is selected from cycloalkyl with up to 10 carbon atoms;

wherein n is 1 or 2; or

Y² is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

These compounds and compositions presented above are not intended to belimiting, but merely representative of the chemical structures andformulas encompassed by the present invention.

Pharmaceutically Acceptable Salts

For the proposed N², N⁴, N⁶-tris(amino-substituted)-1,3,5-triazines, theterms “non-toxic, pharmaceutically acceptable salt” or “pharmaceuticallyacceptable salt” refer to a salt or complex of the 1,3,5-triazinecompounds that retain or enhance the biologically activity of thecompounds described in this invention. Examples of salts are those thatare derived from the interaction of the 1,3,5-triazine compounds orderivatives and an inorganic (mineral acids) or organic acid, as well ascompounds derived from deprotonating an amine nitrogen of the triaminederivatives.

Examples of inorganic acids include, but are not limited to,hydrochloric acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid,nitric acid, nitrous acid, perchloric acid, chloric acid, hypochlorousacid, chlorous acid, phosphoric acid, sulfuric acid, sulfurous acid, andcarbonic acid. Examples of organic acids include, but are not limited toacetic acid, benzene sulfonic acid, benzoic acid, butanoic acid,camphorsulfonic acid, citric acid, ethane sulfonic acid, fumaric acid,glutaric acid, 2-hydroxy acetic acid acids (derivatives where alkylgroup is c=3-7 and hydroxy group is located accordingly), 2-hydroxyalkyl sulfonic acids (derivatives where alkyl group is c=3-7 and hydroxygroup is located accordingly), lactic acid, maleic acid, malic acidmalonic, methane sulfonic acid, naphthalene sulfonic acid, oxalic acid,palmitic acid, propanoic acid, phthalic acid, pyruvic acid, salicylicacid, stearic acid, succinic acid, tartaric acid, p-toluene sulfonicacid, and amino acids (e.g., alanine, N-acetylglycine, arginine,aspartic acid, glutamic acid, glycine, lysine, and phenylalanine).

Examples of salts described here include compounds that derive from adeprotonation reaction of an amine nitrogen of the triamine derivativeswith a strong base, to form an amido salt, compound or complex. Forexample, these compounds include those that are derived from theinteraction or chemical reaction of the 1,3,5-triazine compounds orderivatives acting as a Bronsted or Lewis acid and an inorganic ororganic base to form an ionic and/or complexed species Examples ofinorganic bases, include but not limited to, metallic bases ororganometallic bases such as alkyllithiums or metal hydrides, wherethere is a metallic counterion include, but are not limited to,aluminum, barium, calcium, lithium, magnesium, potassium sodium, andzinc.

Examples of organic bases include, but are not limited to, alky and arylamines as well as ammonia. Included in this description are salts formedfrom the combination or interaction/reaction of inorganic acids (e.g.,Lewis acids) and metallic counterions and the 1,3,4-triazine compoundsor derivatives acting as a Bronsted or Lewis base resulting in theformation of ionic and/or complexed species For all salts and complexesas described above, these are to include hydrated or solvated forms ofthe compounds.

Additionally, this invention also encompasses salts of these triazinederivatives that are non-toxic and pharmaceutically acceptable, such asquaternary ammonium salts, for example [—N⁺R₂R′]X⁻, where the R and R′groups represent hydrogen or an organic group (such as alkyl, alkenyl,alkynyl, aryl, and the like) and the X group is a counter ion (halogen,hydroxide, alkoxide, thioalkoxide, or conjugate base of an organic orinorganic acid). For all salts and complexes as described above, theseare to include hydrated or solvated forms of the compounds.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure Ia:

wherein:R^(1a) is selected from

R^(2a) is selected from

R^(3a) is selected from —H,

R^(4a) is selected from —H or —CH₃;

R^(5a) is selected from

R^(6a) is selected from —H or —CH₃; andR^(7a) is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure IIa:

wherein R^(1a) is selected from:

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure IIIa:

wherein R^(1a) is selected from

R^(2a) is selected from —H or —CH₃.Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure IVa:

wherein:R^(1a) is selected from

R³ is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure Va:

wherein R^(3a) is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure VIa:

wherein R^(3a) is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure VIIa:

wherein R^(1a) is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure VIIIa:

wherein R^(1a) is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure IXa:

wherein:R^(1a) is selected from

R³ is selected from —H,

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure Xa:

wherein R^(3a) is selected from —H,

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XIa:

wherein R^(3a) is selected from —H,

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XIIa:

wherein R^(1a) is selected from

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XIIIa:

wherein R^(1a) is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XIVa:

wherein R^(3a) is selected from —H,

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XVa:

wherein:R^(1a) is selected from

R^(3a) selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XVIa:

wherein R^(3a) is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XVIIa:

wherein:R^(1a) is selected from

R^(5a) is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XVIIIa:

wherein R^(5a) is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XIXa:

wherein R^(5a) is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XXa:

wherein R^(1a) is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XXIa:

wherein R^(1a) is selected from:

R^(4a) is selected from —H or —CH₃.Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XXIIa:

wherein:R^(1a) is selected from

R^(7a) is selected from

Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XXIIIa:

wherein:R^(1a) is selected from

R^(2a) is selected from

R^(3a) is selected from —H,

R^(5a) is selected from,

R^(6a) is selected from —H or —CH₃.Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

In another aspect, compounds and compositions in accordance with thepresent invention comprise analogs of the tris(amino) triazene compoundsof the following general structure XXIVa:

wherein:m is 1 or 0; andR^(3a) is selected from

When m is 0, R^(3a) has a heteroatom bonded to the triazine ring.Compositions comprising compounds of this formula are also encompassedby the present invention, as well as mixtures or combinations ofcompounds of this formula.

Table 1A presents further exemplary compounds of the present invention.The inclusion of compounds in this table is not to be seen as limiting,rather compounds are provided in this table by way of example.

Additionally, compounds in accordance with the present invention includethe compounds listed in Table 1E. Again, these compounds, which arediscussed in the Examples provided herein, are to be considered asexemplary.

TABLE 1E Representative Compounds According to the Present InventionCOMPOUND NUMBER COMPOUND NAME E1  4-Benzyloxy-3-chloro-phenylamine, E2 N-(4-Benzyloxy-3-chloro-phenyl)-N′-cycloheptyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine, E3 N-(4-benzyloxy-3-chloro-phenyl)-N′-cycloheptyl-N″-methyl-N″-piperidin-4-yl-[1,3,5]triazine-2,4,6-triamine, E4 4-[4-Cycloheptylamino-6-(methyl-piperidin-4-yl-amino)-[1,3,5]triazin-2-ylamino]-phenol, E5 4-{4-Cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-1,3,5]triazin-2-ylamino}-phenol, E6 2-Chloro-4-(4,6-dichloro-[1,3,5]triazin-2-ylamino)-phenol, E7 2-Chloro-4-(4-chloro-6-cycloheptylamino-[1,3,5]triazin-2-ylamino)-phenol, E8  N-(1-Benzyl-piperidin-4-yl)-N′-(3-fluoro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-diamine, E9 N-Cycloheptyl-N′-(4-methoxy-phenyl)-N″-piperidin-4-yl-[1,3,5]triazine-2,4,6-triamine, E106-Chloro-N-cyclopropyl-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine, E11N-Cyclopropyl-N′-(3-flouro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine, E12N-Cyclopropyl-N′-(1-ethyl-pyrrolidin-2-ylmethyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine, E136-Chloro-N-(3-chloro-4-methoxy-phenyl)-N′-cyclopropyl-[1,3,5]triazine-2,4-diamine E14N-Cyclopropyl-N′-(3-chloro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine, E156-Chloro-N,N′-bis-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine,E16 N-(1-Ethyl-pyrrolidin-2-ylmethyl)-N′,N″-bis-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine, E171-[4-(3-chloro-4-methoxyanilino)-cycloheptyl amino-1,3,5-triazin-2-yl]-2-azoloamymethanol, E18N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(4-methylpiperzino)-1,3,5-triazine-4,2-diamine, E193-[4-(3-chloro-4-methoxyanilino)-6-cycloheptylamino-1,3,5-triazin-2-yloxy]-2-ethyl-4H-4-pyranone, E201-[3-{4-(3-chloro-4-methoxyanilino)-6-cycloheptylamino-1,3,5-triazine-2-yloxy}piperidino]-1-ethanone, E21N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-isopropoxy-1,3,5-triazine-2,4-diamine, E22N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(2-azolanylmethoxy)-1,3,5-triazine-2,4-diamine, E231-[4-(3-chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazine-2-yl]-piperidin-3-ol], E241-[4-(3-chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazine-2-yl]-piperidin-4-ol], E25N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(1-methyl-2-azolanylmethoxy)-1,3,5-triazine-2,4-diamine, E26N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(1-methyl-4-piperidyloxy)-1,3,5-triazine-2,4-diamine, E27N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(1,4-thiazinan-4-yl)-1,3,5-triazine-4,2-diamine, E28N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(2-fluorophenoxy)-1,3,5-triazine-4,2-diamine, E29N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-[2-(2- fluorophenoxy)ethoxy]-1,3,5-triazine-4,2-diamine, E30N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(6-methyl-2- pyrylmethoxy]-1,3,5-triazine-4,2-diamine, E31N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-N″-methyl-N″- (1-methylpiperidin-4-yl)[1,3,5]triazine-2,4,6-triamine, HCl salt, E326-methoxy-N-(3-Chloro-4-methoxy phenyl)-N′-cyclo heptyl-[1,3,5]triazine-2,4 diamine, E33 6-ethoxy-N-(3-Chloro-4-methoxyphenyl)-N′-cyclo heptyl [1,3,5]triazine-2,4 diamine, E346-isopropoxy-N-(3-Chloro-4-methoxy phenyl)-N′-cyclo heptyl-[1,3,5]triazine-2,4 diamine.III. Antiproliferative Activities

One embodiment of the present invention comprises methods andcompositions comprising the compounds of the present invention for thetreatment and prevention of conditions or diseases that have as anaspect of the disease or condition, unwanted cellular proliferationoccurring or are the result of cellular proliferation. For example, manyvascular diseases, such as cardiovascular diseases, organ transplantsequellae, vascular occlusive conditions including, but not limited to,neointimal hyperplasia, restenosis, transplant vasculopathy, cardiacallograft vasculopathy, atherosclerosis, and arteriosclerosis, arecaused by or have collateral damage due to unwanted cellularproliferation. Smooth muscle cell (SMC) hyperplasia is a major event inthe development of atherosclerosis and is also responsible for thesignificant number of failure rates following vascular procedures suchas angioplasty and coronary artery bypass surgery, particularly due torestenosis. Proliferation of arterial wall SMC in response to localinjury is a major feature of many vascular proliferative disorders.Neointimal hyperplasia is commonly seen after various forms of vascularinjury and a major component of the vein graft's response to harvest andsurgical implantation into high-pressure arterial circulation.Proliferation of SMC in response to local injury is a major feature ofvascular proliferative disorders such as atherosclerosis and restenosisafter angioplasty.

One aspect of the present invention relates to methods and compositionsfor the treatment and prevention of smooth muscle cell (SMC)proliferation, preferably comprising compositions and compounds havingcellular antiproliferative activity. These compounds and compositionscomprising such compounds are referred to as antiproliferative compoundsor compositions. At least one activity of one or more of these compoundsis that the compound has the activity of affecting the synthesis ofproteoglycans including induction and synthesis of proteoglycans andactive fragments of proteoglycans. Thus, one aspect of the activity ofone or more of the compounds and compositions of the present inventioncomprise molecules that induce HSPG production and that regulate SMC(smooth muscle cell) proliferation.

Compounds of the present invention that have at least the activity ofaffecting cellular proliferation are shown in TABLE 3. The compoundsshown in this Table have the activity of affecting cellularproliferation as measured by the assays taught herein. The inclusion ofcompounds in the categories of the Tables disclosed herein are not to beseen as limiting, in that compounds included in such Tables have atleast the activity shown for inclusion in the Table and may have more orother activities. Nor are the Tables to be seen as limiting in thatthese are the only compounds disclosed herein that have that activity,representative compounds are shown in the Tables that have at least thatparticular activity for inclusion in the Table. One or more compoundsdisclosed herein have at least an activity that has utility in treatmentof disease states.

Examples of compounds that show at least this activity and utility areshown in the following structure:

or an ene, a diene, a triene, or an yne derivative thereof; a saturatedderivative thereof; a stereoisomer thereof; or a salt thereof;wherein:

R¹ is in each occurrence independently selected from —H; linear orbranched alkyl with up to 10 carbon atoms; or cycloalkyl with up to 10carbon atoms;

X¹ is selected from m-F, m-Cl, m-Br, m-I, m-CN, m-NO₂, m-SO₂R¹, orm-SO₂OR¹, or X¹ and X² together is a fused benzene, pyridine, or dioxanering;

X² is selected from p-OR¹, p-SR¹, p-NR¹ ₂, p-OM, or p-SM, wherein M isselected from Li, Na, K, Mg, or Ca;

Y¹ is selected from cycloalkyl with up to 10 carbon atoms; linear orbranched alkyl with up to 10 carbon atoms; CH₂R², wherein R² is acycloalkyl with up to 10 carbon atoms; or

wherein n is 1 or 2;

AY² is selected from a halogen or OR¹, or

A is NR¹ and Y² is selected from R¹,

Further examples of compounds that show at least this activity andutility are presented in Table 3, where compound activity is alsopresented. The activity scale used in Table 3 as follows (numbers areinclusive): “+++” represents IC₅₀ of less than about 3 μM; “++”represents IC₅₀ of between about 3 and about 7 μM; and “+” representsIC₅₀ of greater than about 7 μM. In addition, compounds, to includecompositions thereof, encompassed within the scope of structuresIa-XXIVa, respectively, or listed in Tables 1A and 1E may be likewiseemployed in this embodiment and/or aspect of the present invention.Further, any hydrogen atoms that are required for any atom to attain itsusual valence in a structure presented in Table 3, whether a carbon atomor a heteroatom, should be inferred if it is not specifically indicated.

In addition to the above compounds, the following compounds andcompositions comprising these compounds are active in ananti-proliferation assay (Perlecan). These compounds and compositionscomprising these compounds are, among other things, generally useful fortreating cardiovascular disorders associated with proliferativeactivity. Specifically, these compounds includeN²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine,andN²-cycloheptyl-N⁴-methyl-N⁴-(1-methyl-piperidin-4-yl)-N⁶-naphthalen-2-yl-1,3,5-triazine-2,4,6-triamine.Using the same activity scale used in Table 3, and discussed above, thefirst compound,N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine,is characterized as a compound exhibiting medium or moderate activity,while the second compound,N²-cycloheptyl-N⁴-methyl-N⁴-(1-methyl-piperidin-4-yl)-N⁶-naphthalen-2-yl-1,3,5-triazine-2,4,6-triamine,is characterized as a compound exhibiting high activity.

As used herein, when a proteoglycan is referred to, the entire moleculeor fragments are included therein. For example, perlecan refers to theentire perlecan molecule or fragments thereof. Different fragments ofperlecan may have the same or different effects on cells and the effectsmay be the same as or different from the effects that the entireperlecan molecule has on cells. These fragments and activities arecontemplated in the present invention and compounds included in thepresent invention may have at least one activity that modulates oraffects the fragements' activities or the entire molecule's activities.Although the discussion herein refers specifically to perlecans it isimportant to note that the compositions, methods, and assays describedherein are equally applicable in the context of other proteoglycans,including HSPGs, and including but not limited to, chondroitan sulfates(e.g., A, B, and C), dermatan sulfates, syndecans and glypicans.

Methods for identifying the activity and screening for one or more ofthese compounds or molecules that induce synthesis of proteoglycans suchas HSPG (heparan sulfate proteoglycan) are taught in U.S. patentapplication Ser. No. 10/091,357, which is incorporated herein in itsentirety. Assays of effects of compounds in vivo are also taught in theincorporated references and are known to those skilled in the art. Ingeneral, methods comprise the addition of such compounds to assays andmeasurement of HSPG synthesis including, but not limited to, theproduction of syndecans, glypicans and perlecans, for example, syndecans1, 2 and 4; and glypican-1. Other assays that can be used to determinethe activity of the compounds of the present invention include othermethods for measuring the induction of perlecan synthesis. For example,in one assay, perlecan is induced in cells by certain inducers, and theresponse is measured. Compounds of the present invention are then addedto a replicate assay and the effect on perlecan induction is determined.Using such methods, compounds are determined that can either inhibitperlecan, elevate induction of perlecan, or that have no effect at all.Those compounds that are effective as therapeutic agents can then beused in animals, humans or patients with cellular proliferation diseaseaspects, such as vascular-associated diseases or SMC proliferationpathologies.

Another assay for determining compounds having SMC effects comprisesadding a composition suspected of affecting SMC proliferation to smoothmuscle cells in growth medium or serum-free medium. The change in cellproliferation can be measured by methods known to those skilled in theart, such as incorporation of labeled nucleotides into dividing cells'DNA, and compared to the proliferation of cells which are not treatedwith the compound. Other measurements include directly determininglevels of HSPG synthesis by measuring the amount or change in amount ofHSPG such as with ELISA for HSPGs, and compared to the amount of HSPGsynthesis in untreated cells. Other indirect or direct measurement arecontemplated by the present invention and are known to those skilled inthe art. For example, such methods include, but are not limited to,measurement of RNA levels, RT-PCR, Northern blotting, Western blottingpromoter-based assays to identify compounds that affect one or moreproteoglycans and assays for proteoglycan biological activity shown byrecombinant proteins, partially purified proteins, or lysates from cellsexpressing proteoglycans in the presence or absence of compounds ofinterest.

An assay for identifying and determining an activity of one or more ofthe compounds of the present invention comprises identifying compoundsthat interact with the promoter regions of a gene, or interact andaffect proteins that interact with the promoter region, and areimportant in the transcriptional regulation of the protein's expression.For example, if perlecan were the protein, in general, the methodcomprises a vector comprising regulatory sequences of the perlecan geneand an indicator region controlled by the regulatory sequences, such asan enzyme, in a promoter-reporter construct. The protein product of theindicator region is referred to herein as a reporter enzyme or reporterprotein. The regulatory region of the sequence of perlecan comprises arange of nucleotides from approximately −4000 to +2000 wherein thetranscription initiation site is +1, more preferably, from −2500 to+1200, most preferably, from −1500 to +800 relative to the transcriptioninitiation site.

Cells are transfected with a vector comprising the promoter-reporterconstruct and then treated with one or more compositions comprising atleast one compound of the present invention. For example, thetransfected cells are treated with a composition comprising a compoundsuspected of affecting the transcription of perlecan and the level ofactivity of the perlecan regulatory sequences are compared to the levelof activity in cells that were not treated with the compound. The levelof activity of the perlecan regulatory sequences are determined bymeasuring the amount of the reporter protein or determining the activityof the reporter enzyme controlled by the regulatory sequences. Anincrease in the amount of the reporter protein or the reporter enzymeactivity shows a stimulatory effect on perlecan, by positively affectingthe promoter, whereas a decrease in the amount or the reporter proteinor the reporter enzyme activity shows a negative effect on the promoterand thus, on perlecan.

Additionally, the present invention comprises methods and compositionsthat can be used with gene therapy methods and composition, such asthose gene therapy methods comprising administering compositionscomprising nucleic acids that affect the synthesis or expression ofHSPGs, particularly perlecan. Such methods and compositions are taughtin U.S. patent application Ser. No. 10/091,357, incorporated herein byreference.

The present invention comprises methods and compositions for mediatingproteoglycan synthesis, expression and for the maintenance of SMC in aquiescent state. Methods and compositions of the present inventioncomprise treatment and prevention of vascular diseases and pathologiesrelated to celluar proliferation, such as SMC proliferation. Suchmethods and compositions comprise methods for inhibition of smoothmuscle cell (SMC) growth and proliferation, and for induction ofquiescence in smooth muscle cells. Embodiments of the present inventioncomprise methods and compositions for inducing proteoglycan synthesis,particularly HSPG synthesis and expression including, but not limitedto, the induction of HSPGs such as syndecans, glypicans and perlecans,and preferably perlecan synthesis and gene expression. Perlecan is amajor extracellular HSPG in the blood vessel matrix. It interacts withextracellular matrix proteins, growth factors and receptors. Perlecan isalso present in basement membranes other than blood vessels and in otherextracellular matrix structures.

The activities of the compounds included in the present invention affectcells or tissues to increase the synthesis of proteoglycans by thosecells or tissues or may act directly upon one or more proteoglycans tomodulate the biological activity or to increase the biological stabilityof the proteoglycan itself, for example, of the protein perlecan.Activities also included herein are ones that increase the biosynthesisof one or more proteoglycans by increasing the transcription of thepoteoglycan gene, increasing the biological stability of theproteoglycan mRNA or increasing the translation of proteoglycan mRNAinto protein. Further activites include activities of compounds that canblock or decrease the effects of agents or proteins that inhibit theactivity of proteoglycans.

The present invention comprises methods and compositions for thetreatment and prevention of smooth muscle cell proliferation, includingvascular occlusive pathologies. Such methods comprise administration ofcompositions comprising compounds capable of inhibiting SMCproliferation, such as compositions comprising compounds disclosedherein that inhibit SMC proliferation. Administration of such compoundsthat are effective in inhibiting SMC proliferation are administered tohumans and animals suspected of having or who have, for example,vasculopathy or who have undergone angioplasty or other proceduresdamaging to the endothelium. Effective amounts are administered to suchhumans and animals in dosages that are safe and effective, including,but not limited to, the ranges taught herein. Routes of administrationinclude, but are not limited to, those disclosed herein. As disclosedherein, compositions comprising such compounds may be used inconjunction with other therapeutic agents or in methods comprising stepssuch as altered patient activities, including, but not limited to,changes in exercise or diet.

The compounds of the present invention are useful in the treatment orprophylaxis of at least one cardiovascular disease in a cell, tissue,organ, animal, or patient including, but not limited to, vascularocclusive lesions including atherosclerosis, transplant vasculopathy,cardiac allograft vasculopathy, restenosis, graft atherosclerosis aftercoronary transplantation, cardiac stun syndrome, myocardial infarction,congestive heart failure, stroke, ischemic stroke, hemorrhage,arteriosclerosis, atherosclerosis, restenosis, diabetic aterioscleroticdisease, hypertension, arterial hypertension, renovascular hypertension,syncope, shock, syphilis of the cardiovascular system, heart failure,cor pulmonale, primary pulmonary hypertension, cardiac arrhythmias,atrial ectopic beats, atrial flutter, atrial fibrillation (sustained orparoxysmal), post perfusion syndrome, cardiopulmonary bypassinflammation response, chaotic or multifocal atrial tachycardia, regularnarrow QRS tachycardia, specific arrythmias, ventricular fibrillation,His bundle arrythmias, atrioventricular block, bundle branch block,myocardial ischemic disorders, coronary artery disease, angina pectoris,myocardial infarction, cardiomyopathy, dilated congestivecardiomyopathy, restrictive cardiomyopathy, valvular heart diseases,endocarditis, pericardial disease, cardiac tumors, aordic and peripheralaneuryisms, aortic dissection, inflammation of the aorta, occulsion ofthe abdominal aorta and its branches, peripheral vascular disorders,occulsive arterial disorders, peripheral atherlosclerotic disease,thromboangitis obliterans, functional peripheral arterial disorders,Raynaud's phenomenon and disease, acrocyanosis, erythromelalgia, venousdiseases, venous thrombosis, varicose veins, arteriovenous fistula,lymphederma, lipedema, unstable angina, reperfusion injury, post pumpsyndrome, ischemia-reperfusion injury, and the like. Such methods canoptionally comprise administering an effective amount of a compositionor pharmaceutical composition comprising at least one compound to acell, tissue, organ, animal or patient in need of such modulation,treatment or therapy.

Proteoglycan-associated diseases that are treatable with the compoundsof the present invention include, but are not limited to, hereditarymultiple exostosis, mucopolysaccharidosis types I-III and VII, commonlyknown as Hurler's Syndrome, Hunter's Syndrome, Sanfilippo's Syndrome andSly's Syndrome respectively, Alzheimer's disease, Simpson-Golabi-Behmelsyndrome, fibroblast growth factor related disorders, herpes simplexvirus, dengue fever, Parkinson's disease, renal disease, musculardystrophy, Schwarts-Jampel syndrome, proteinuric glomerulopathies,myotonia and skeletal dysplasia, kyphoscoliosis, dyssegmental dysplasia,Silverman-Handmaker type, chondrodysplasia, periodontitis, rheumatoidand osteoarthritis, Gerstmann-Straussler syndrome, Creutzfeldt-Jakobdisease, scrapie, carcinomas, Happle syndrome, macular dystrophy, bonediseases, corneal diseases, leukocyte-mediated disease, collagen fibrilassembly disorder and coronary heart disease and other vasculardisorders.

IV. Glycosidase Modulation Activity

The present invention also comprises methods and compositions comprisingcompounds described herein that have an activity associated withmodulation of glycosidase enzymes and thus, affecting the substrates forsuch enzymes. Glycosidase enzymes and their activity with theirsubstrates, such as proteoglycans or glycated proteins, are aspects of avariety of diseases such as vascular conditions, including thoseconditions discussed supra, proteoglycan-associated diseases, supra,associated diseases with vascular components, including but not limitedto, kidney disease, ischemic heart disease, cardiovascular disease,generalized vascular disease, proliferative retinopathy, andmacroangeopathy, inflammatory diseases and metastatic diseases such ascancer, cellular proliferative conditions, and solid and blood bornetumors or other oncological conditions. Compounds described herein thathave an activity that affects the concentrations of substrates ofglycosidase enzymes are used in methods of treatment of such vascular,inflammatory, metastatic and systemic diseases.

An aspect of the present invention comprises methods and compositionsfor the modulation of enzymes, such as glycosaminoglycan degradingenzymes, which affect or are affected by proteoglycan levels, amount oractivity. For example, the present invention comprises methods andcompositions comprising compounds that modulate enzymes including butnot limited to, heparanase, chondroitanase, heparan sulfateendoglycosidase, heparan sulfate exoglycosidase, polysaccharide lyases,keratinase, hyauronidase, glucanase, amylase, glycosidases, or otherproteoglycan degrading enzymes are useful for the treatment ofconditions such as diabetic vasculopathy, cancer, inflammatory diseases,autoimmune diseases and cardiovascular diseases. For example, thepresent invention comprises methods and compositions of compounds thatinhibit, impair or down-regulate the activity of proteoglycan degradingenzymes.

Proteoglycans such as HSPG are important components of thesubendothelial extracellular matrix and the basement membrane of bloodvessels. Rosenberg et al., 99 J. CLIN. INVEST. 2062-70 (1997). Basementmembranes are continuous sheets of extracellular matrix composed ofcollagenous and noncollagenous proteins and proteoglycans that separateparenchymal cells from underlying interstitial connective tissue. Theyhave characteristic permeabilities and play a role in maintaining tissuearchitecture.

In addition to HSPGs, the basal lamina consists predominantly of acomplex network of adhesion proteins, fibronectin, laminin, collagen andvitronectin. Wight et al., 6 CURR. OPIN. LIPIDOL. 326-334 (1995).Heparan sulfate (HS) is an important structural component of the basallamina. Each of the adhesion proteins interacts with HS side chains ofHSPGs within the matrix. Thus, HSPGs function as a barrier to theextravasation of metastatic and inflammatory cells. Cleavage of HS bythe endoglycosidase heparanase produced by metastatic tumor cells andinflammatory cells destroys the filtering properties of the lamina. Inaddition, the degradation of the HS may assist in the disassembly of theextracellular matrix and thereby facilitate cell migration by allowingblood bome cells to escape into the bloodstream. Vlodavsky et al., 12INVASION METASTASIS 112-127 (1992).

Heparanase activity has been described in a number of tissues and celltypes including liver, placenta, platelets, fibroblasts, neutrophils,activated T and B-lymphocytes, monocytes, and endothelial cells (7-16).Nakajima et al., (31) CANCER LETT. 277-283 (1986); Nakajima et al., 36J. CELL. BIOCHEM. 157-167 (1988); Ricoveri et al., 46 CANCER RES.3855-3861 (1986); Gallagher et al., 250 BIOCHEM. J. 719-726 (1988);Dempsey et al., 10 GLYCOBIOLOGY 467 (2000); Goshen et al., 2 MOL. HUM.REPROD. 679 (1996); Parish et al., 76 IMMUNOL CELL BIOL. 104-113 (1998);Gilat et al., 181 J. EXP. MED. 1929-1934 (1995); Graham, et al., 39BIOCHEM. MOL. BIOL. INT. 56371 (1996); Pillarisetti et al., 270 J. BIOL.CHEM. 29760-29765 (1995). An important process in tissue invasion byblood-borne tumor cells and white cells involves their passage throughthe vascular endothelial cell layer and subsequent degradation of theunderlying basal lamina or basement membranes and extracellular matrixwith a battery of secreted proteases and glycosidases. Nakajinia et al.,220 SCIENCE 611-613 (1983); Vlodavsky et al., 12 INVASION METASTASIS112-127 (1992).

Heparanase activity was shown to correlate with the metastatic potentialof animal and human tumor cell lines. Nakajima et al., 31 CANCER LETT.277-283 (1986); Nakajima et al., 212 PROG CLIN BIOL RES. 113-122 (1986);Freeman et al., 325 BIOCHEM. J. 229-237 (1997); Vlodavsky et al., 5 NAT.MED. 793-802 (1999); Hulett et al., 5 NAT MED. 803-809 (1999). It isalso known to regulate growth factor activity. Many growth factorsremain bound to heparan sulfate in storage form and are disassociated byheparanase during angiogenesis, improving the survival rate of cancercells.

Serum heparanase levels in rats were higher by more than an order ofmagnitude after injection of the rats with highly metastatic mammaryadenocarcinoma cells. In addition, heparanase activity in the sera ofrats bearing MTLn3 tumors correlated well with the extent of themetastases. Moreover, serum/urine heparanase activity in cancer patientswas shown to be 2-4 fold increased in particular where tissue metastaseswere present. Because the cleavage of HS appears to be essential for thepassage of metastatic tumor cells and leukocytes through basementmembranes, studies of heparanase inhibitors provides the potential ofdeveloping a novel and highly selective class of anti-metastatic andanti-inflammatory drugs.

The present invention comprises methods and compositions comprisingcompounds that modulate heparanase activity or the activity of otherglycosidases, including, but not limited to enzymes withglycosaminoglycan activity such as chondroitinase, heparan sulfateendoglycosidase, heparan sulfate exoglycosidase, polysaccharide lyases,keratinase, hyaluranidase, glucanase, and amylase. Compounds of thepresent invention that have at least the activity of modulatingglycosidase enzyme activity are shown in TABLE 6. The compounds shown inthis Table have the activity of modulating glycosidase enzyme activityas measured by the assays taught herein. The inclusion of compounds inthe categories of the Tables disclosed herein are not to be seen aslimiting, in that compounds included in such Tables have at least theactivity shown for inclusion in the Table and may have more or otheractivities. Nor are the Tables to be seen as limiting in that these arethe only compounds disclosed herein that have that activity,representative compounds are shown in the Tables that have at least thatparticular activity for inclusion in the Table. One or more compoundsdisclosed herein have at least an activity that has utility in treatmentof disease states.

Examples of compounds that show at least this activity and utility areshown in the following formula:

or an ene, a diene, a triene, or an yne derivative thereof; a saturatedderivative thereof; a stereoisomer thereof; or a salt thereof;wherein:

R¹ is in each occurrence independently selected from —H; linear orbranched alkyl with up to 10 carbon atoms; or cycloalkyl with up to 10carbon atoms;

X¹ is selected from H, m-F, m-Cl, m-Br, m-I, m-CN, m-NO₂, M-SO₂R¹, orm-SO₂OR¹;

X² is selected from o-R¹, p-OR¹, p-SR¹, p-NR¹ ₂, p-OM, or p-SM, whereinM is selected from Li, Na, K, Mg, or Ca;

Y¹ is selected from cycloalkyl with up to 10 carbon atoms or

Y² is selected from linear or branched alkyl with up to 10 carbon atoms,cycloalkyl with up to 10 carbon atoms, or

and R² is —H; or NY²R² together is selected from

wherein x is an integer from 3 to 5,

wherein q is an integer from 0 to 6, or

wherein Z² is selected from R¹ or

Further examples of compounds that show at least this activity andutility are presented in Table 6, where compound activity is also shown.The activity scale used in Table 6 is as follows (numbers areinclusive): “+++” represents between about 70 and about 100% inhibition;“++” represents between about 30 and about 40% inhibition; and “+”indicates between 0 and about 30% inhibition, all at 5 μM compoundconcentration. In addition, compounds, to include compositions thereof,encompassed within the scope of structures Ia-XXIVa, respectively, orlisted in Tables 1A and 1E may be likewise employed in this embodimentand/or aspect of the present invention. Also note that any hydrogenatoms that are required for any atom to attain its usual valence in astructure presented in Table 6, whether a carbon atom or a heteroatom,should be inferred if it is not specifically indicated.

Compounds or compositions comprising such compounds that are effectivein modulating glycosidase enzyme activity are useful in treating and/orpreventing cancer including, but not limited to, malignant andnon-malignant cell growth, leukemia, acute leukemia, acute lymphoblasticleukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML),chromic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL),hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma,Hodgkin's disease, a malignamt lymphoma, non-hodgkin's lymphoma,Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectalcarcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignanthistiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy,solid tumors, adenocarcinomas, sarcomas, malignant melanoma, hemangioma,metastatic disease, cancer related bone resorption, cancer related bonepain, and the like.

In another aspect of the present invention, the compounds disclosedherein are useful in modulating heparanase activity or the activity ofother glycosidases as a means for treating and preventing autoimmunediseases. Generally autoimmune disease results when (1) the immunesystem mistakenly identifies a cell surface molecule on normal tissue asa foreign molecule (2) the synthesis and secretion of chemokines,cytokines and lymphokines is not shut down after the eradication of thedisease or (3) the immune system overreacts to the apparent infectionand destroys vast quantities of surrounding normal tissue.

To be effective in an immune response, the immune effector cells mustbind to the luminal/apical surface of the blood vessel walls. This isaccomplished through the interaction of adhesion molecules on the immuneeffector cells with their locally upregulated cognate receptors on theendothelial cells lining the vasculature near the site of infection.After binding to the apical surface and before entering the inflamedtissue, the immune effector cells must breach the basement membrane (BM)and extracellular matrix (ECM) that surround the basal portion of theblood vessels and give the vessels their shape and strength. The BM andECM consists of structural proteins embedded in a fiber meshworkconsisting mainly of complex carbohydrate containing structures(glycosaminoglycans), of which the main constituent is heparin sulfateproteoglycan (HSPG). In order to breach this barrier the immune effectorcell must weaken or destroy it, which is accomplished through the localsecretion of proteases and heparanase(s).

Thus, the inhibition of heparanase or the activity of other glycosidasesusing the compounds of the present invention finds utitlity in treatingarthritis and other autoimmune diseases. More specifically, thecompounds of the present invention are useful in the treatment orprophylaxis of at least one autoimmune-related disease in a cell,tissue, organ, animal, or patient including, but not limited to,rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onsetjuvenile rheumatoid arthritis, psoriatic arthritis, ankylosingspondilitis, gastric ulcer, seronegative arthropathies, osteoarthritis,inflammatory bowel disease, ulcerative colitis, systemic lupuserythematosis, antiphospholipid syndrome, iridocyclitis/uveitis/opticneuritis, idiopathic pulmonary fibrosis, systemic vasculitis/wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergiccontact dermatitis, allergic conjunctivitis, hypersensitivitypneumonitis, transplants, organ transplant rejection, graft-versus-hostdisease, systemic inflammatory response syndrome, sepsis syndrome, grampositive sepsis, gram negative sepsis, culture negative sepsis, fungalsepsis, neutropenic fever, urosepsis, meningococcemia,trauma/hemorrhage, burns, ionizing radiation exposure, acutepancreatitis, adult respiratory distress syndrome, rheumatoid arthritis,alcohol-induced hepatitis, chronic inflammatory pathologies, Crohn'spathology, sickle cell anemia, diabetes, nephrosis, atopic diseases,hypersensitity reactions, allergic rhinitis, hay fever, perennialrhinitis, conjunctivitis, endometriosis, asthma, urticaria, systemicanaphalaxis, dermatitis, pernicious anemia, hemolytic disesease,thrombocytopenia, graft rejection of any organ or tissue, kidneytranslplant rejection, heart transplant rejection, liver transplantrejection, pancreas transplant rejection, lung transplant rejection,bone marrow transplant (BMT) rejection, skin allograft rejection,cartilage transplant rejection, bone graft rejection, small boweltransplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, -meditated cytotoxicity, type III hypersensitivityreactions, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy,monoclonal gammopathy, and skin changes syndrome), polyneuropathy,organomegaly, endocrinopathy, monoclonal gammopathy, skin changessyndrome, anti-phospholipid syndrome, pemphigus, scleroderma, mixedconnective tissue disease, idiopathic Addison's disease, autoimmunehemolytic anemia, autoimmune hepatitis, idiopathic pulmonary fibrosis,scleroderma, diabetes mellitus, chronic active hepatitis, vitiligo,vasculitis, post-MI cardiotomy syndrome, type IV hypersensitivity,contact dermatitis, hypersensitivity pneumonitis, allograft rejection,granulomas due to intracellular organisms, drug sensitivity,metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, ankylosingspondylitis, Behcet's disease, bullous pemphigoid, cardiomyopathy,celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, coldagglutinin disease, discoid lupus, essential mixed cryoglobulinemia,fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré, Hashimoto'sthyroiditis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy,insulin dependent diabetes, juvenile arthritis, lichen planus, ménière'sdisease, multiple sclerosis, pemphigus vulgaris, polyarteritis nodosa,Cogan's syndrome, polychondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumaticfever, Sjögren's syndrome, stiff-man syndrome, Takayasu arteritis,temporal arteritis/giant cell arteritis, Wegener's granulomatosis; okt3therapy, anti-cd3 therapy, cytokine therapy, chemotherapy, radiationtherapy (e.g., including but not limited toasthenia, anemia, cachexia,and the like), chronic salicylate intoxication, and the like.

Compounds having heparanase activity inhibition, that are effective forexample, in treatment of cancer and autoimmune disease, can bedetermined using assays such as those disclosed in U.S. patentapplication Ser. No. 09/952,648, which is incorporated herein in itsentirety. Such assays, which are used for measurement of cellular andenzymatic activities, both qualitatively and quantitatively, and inmethods for diagnosing metastases, metastatic potential and inflammatorystates, are performed with and without the addition of at least one ofthe compounds of the present invention to determine the activity of thecompound. Existing heparanase assays are taught in Goshen et al., 2 MOL.HUM. REPROD. 679-84 (1996); Nakajima et al., 31 CANCER LETT. 277-83(1986); and Vlodasky et al., 12 INVASION METASTASIS 112-27 (1992);Freeman and Parish, 325 BIOCHEM. J. 229-37 (1997); Kahn and Newman, 196ANAL. BIOCHEM. 373-76 (1991). Solid-phase heparanase assays have alsobeen developed where chemically and biosynthetically radiolabeledheparin and HS chains were attached to a solid support, with release ofradiolabel from the solid support being a measure of enzyme activity.Assays using such procedures are taught in U.S. Pat. No. 4,859,581,which is entirely expressly herein incorporated by reference.

In general, a preferred assay comprises attaching one of a bindingpartner to a substrate for the enzyme to be measured, forming thesubstrate-binding partner. Incubation with a sample comprising theenzyme to be measured allows for activity by the enzyme to be measuredin a reaction mixture. A portion or the whole reaction mixture,depending on the amount needed, is then mixed with the complementarybinding partner, so that the binding partners are bound together. Thisis the first binding reaction. After incubating to allow for binding,washings are performed. A complementary binding partner, complementaryto the first binding partner attached to the substrate, is added. Thiscomplementary binding partner may or may not be the same as the firstcomplementary binding partner. This is the second binding reaction. Thecomplementary binding partner in the second binding reaction is labeledin a manner that is detectable. For example, the complementary bindingpartner is labeled with an enzyme that causes a detectable color changewhen the appropriate reaction conditions exist. The difference betweenthe activity of the enzyme in the presence of a compound and the absenceof compound is used to determine the activity of the compound.

An example of a heparanase assay comprises the following steps. Acomposition comprising biotin-HS (heparan sulfate) is mixed with abiological sample such as a tumor sample, bodily fluid, or other fluidsuspected of having heparanase activity, to form a reaction mixture.This sample may be pretreated to remove contaminating or reactivesubstances such as endogenous biotin. A control portion for thisreaction mixture does not contain a compound of the present invention,whereas a test portion contains one or more compounds disclosed herein.After incubation, an aliquot or portion of the reaction mixture portionsis removed and placed in a biotin-binding plate. The biotin-bindingplate comprises any means for binding biotin, preferably to a solidsurface. See WO 02/23197, which is entirely expressly incorporatedherein by reference. After washing with buffers, a streptavidin-enzymeconjugate is added to the biotin-binding plate. Reagents for the enzymeare added to form a detectable color product. For example, a decrease incolor formation, from a known standard, indicates there was heparanaseactivity in the sample. The difference between the activity of theenzyme in the presence of a compound and the absence of compound is usedto determine the activity of the compound.

Using the above assays or those taught in the Examples herein, theamount of enzyme activity in a sample can be determined and theactivities of compounds of the present invention can be determined. Forexample, a composition comprising a compound of the present invention isadded to a known amount of heparanase either before or during theincubation of the heparanase and its substrate-binding partner. If thecompound alters the activity of the heparanase, the assay methods of thepresent invention will show a change in the amount of detectable label.Such assays are used for high throughput determination of the activityof compounds. See WO 02/23197, which is entirely expressly incorporatedherein by reference.

The activities of the compounds included in the present inventionmodulate the activity of glycosidases, either positively or negatively,include effects on the glycosidases either directly or indirectly. Thecompounds may modulate the synthesis of glycosidases by cells or tissuesor may act directly upon one or more glycosidases to modulate thebiological activity or the biological stability of the enzyme itself,for example, heparanase. Activities also included herein are ones thatincrease the biosynthesis of one or more glycosidases by increasing thetranscription of the glycosidase gene, increasing the biologicalstability of the glycosidase mRNA or increasing the translation ofglycosidase mRNA into protein. Further activites include activities ofcompounds that can block or decrease the effects of agents or proteinsthat inhibit the activity of glycosidases. Additionally, acitivities areincluded that affect the substrates for the glycosidases, such as thosediscussed supra in relation to proteoglycans, or affect the bindingparameters of the enzyme with its substrate, cofactors or stimulatory orinhibitory factors.

The present invention comprises methods and compositions for thetreatment and prevention of diseases or conditions that present orresult from glycosidase activity. Such methods comprise administrationof compositions comprising compounds capable of modulating heparanaseactivity, such as compositions comprising compounds disclosed hereinthat inhibit heparanase activity. Administration of such compounds thatare effective in modulating heparanase activity are administered tohumans and animals suspected of having or who have, for example,inflammatory conditions, autoimmune disease or diabetic vasculopathy.Effective amounts are administered to such humans and animals in dosagesthat are safe and effective, including, but not limited to, the rangestaught herein. Routes of administration include, but are not limited to,those disclosed herein. As disclosed herein, compositions comprisingsuch compounds may be used in conjunction with other therapeutic agentsor in methods comprising steps such as altered patient activities.

V. Inflammation Modulation

An embodiment of the present invention comprises methods andcompositions comprising compounds of the present invention for thetreatment and prevention of conditions or diseases that have as anaspect of the disease or condition, inflammation. An aspect of thepresent invention is directed to methods and compositions comprisingcompounds that are effective in inhibiting inflammation, particularlyinflammation associated with the accumulation or presence of glycatedproteins or AGE. The activity of modulating inflammation includes, butis not limited to, inhibiting inflammation and/or its associated cellactivation by glycated proteins or AGE, blocking the glycation ofproteins, blocking AGE interactions with receptors, blocking AGE-inducedsignaling or signaling-associated inflammatory responses, cytokineinduction, synthesis or release, AGE formation or AGE cross-linking.

The present invention also provides compositions for and methods oftreatment of biological conditions including, but not limited to,vascular complications of type I and type II diabetic-inducedvasculopathies, other vasculopathies, microangiopathies, renalinsufficiency, Alzheimer's syndrome, and inflammation-induced diseasessuch as atherosclerosis. Other inflammatory related diseases include,but are not limited to, inflammatory diseases of the joint such asrheumatoid arthritis, osteoarthritis, autoimmune diseases such as thosetaught supra, streptococcal cell-wall induced arthritis,adjuvant-induced arthritis, bursitis; inflammatory diseases of thethyroid such as acute, subacute and chronic thyroiditis, pelvicinflammatory disease, hepatitis; inflammatory bowel diseases such asCrohn's disease and colitis; neuroinflammatory diseases such as multiplesclerosis, abscess, meningitis, encephalitis, and vasculitis;inflammatory diseases of the heart such as myocarditis, chronicobstructive pulmonary disease, atherosclerosis, pericarditis;inflammatory diseases of the skin such as acute inflammatory dermatoses(urticaria (hives), spongiotic dermatitis, erythema multiforme (emminor), Stevens-Johnson syndrome (sjs, em major), toxic epidermalnecrolysis (ten) and chronic inflammatory dermatoses (psoriasis, lichenplanus, discoid lupus erythematosus, acne vulgaris); inflammatorydiseases of the eye such as uveitis, allergic conjunctivitis, cornealinflammation, intraocular inflammation, iritis; laryngitis and asthma.

The compounds of the present invention have utility in inhibitinginflammation and/or its associated cell activation by glycated proteinsor AGE. Pharmacological inhibition of AGE-induced cell activationprovides the basis for therapeutic intervention in many diseases,notably in diabetic complications and Alzheimer's disease. Therapeuticapproaches for inhibition of AGE-induced inflammation include, but arenot limited to, blocking the glycation of proteins, blocking AGEinteractions with receptors and blocking AGE-induced signaling orsignaling-associated inflammatory responses.

At least one activity of some of the compounds of the present inventionis to block AGE effects by inhibiting AGE-induced signaling. Thesequence of these signaling events leading to inflammation are notclear, but inhibition of these signaling events leads to reduced or noinflammatory results. Compounds that block AGE-induced up-regulation ofinflammatory molecules were determined using screening assays. Otheraspects of the present invention comprise methods and compositionscomprising compounds that block glycated protein-induced inflammation.Some compounds may affect AGE formation or AGE cross-linking.

At least one activity of some of the compounds of the present inventionis to block AGE effects by inhibiting reactions with receptors of AGEand such activities are also contemplated by the methods of the presentinvention for treatment of related pathologies. For example, RAGE, aknown receptor for AGE, is a therapeutic target. Blocking RAGE inhibitedAGE-induced inflammation. Prior to use of the compounds of the presentinvention, the multiple functions of RAGE and possible long term sideeffects of accumulated AGE in plasma, have prevented this method oftreatment from being implemented. However, using the methods andcompositions of the present invention, more specific inhibitorycompounds can be used for treatments and overcome the current problemswith treatments that target receptors.

Compounds of the present invention that have at least the activity ofmodulating inflammation activity are shown in TABLE 5. The compoundsshown in this Table have the activity of modulating inflammationactivity as measured by the assays taught herein. The inclusion ofcompounds in the categories of the Tables disclosed herein are not to beseen as limiting, in that compounds included in such Tables have atleast the activity shown for inclusion in the Table and may have more orother activities. Nor are the Tables to be seen as limiting in thatthese are the only compounds disclosed herein that have that activity,representative compounds are shown in the Tables that have at least thatparticular activity for inclusion in the Table. One or more compoundsdisclosed herein have at least an activity that has utility in treatmentof disease states.

Examples of compounds that show at least this activity and utility areshown in the following formula:

or an ene, a diene, a triene, or an yne derivative thereof; a saturatedderivative thereof; a stereoisomer thereof; or a salt thereof;wherein:

R¹ is in each occurrence independently selected from —H; linear orbranched alkyl with up to 10 carbon atoms; cycloalkyl with up to 10carbon atoms; aryl; or (CH₂)_(x)CN, wherein x is an integer from 0 to 6;

E is CH or N;

n is an integer from 0 to 3;

X¹ is selected from —H, m-F, m-Cl, m-Br, m-I, m-CN, m-NO₂, M-SO₂R¹,m-SO₂OR¹, m-NC(O)R¹, or o-F, or X¹ and X² together is a fused benzene,pyridine, or dioxane ring;

X² is selected from —H, o-Cl, o-Br, o-CF₃, o-R¹, p-OR¹, p-SR¹, p-NR¹ ₂,p-F, p-Cl, p-Br, p-CF₃, p-CN, p-C(O)OR¹, p-NC(O)R¹, p-(4-morpholinyl),or p-(4-methyl-1-piperizinyl);

AY¹ is a halogen, or A is NR¹ or O and Y¹ is selected from cycloalkylwith up to 10 carbon atoms, cycloalkyl with up to 10 carbon atomssubstituted with R¹, linear or branched alkyl with up to 10 carbonatoms, CH₂R₁, (CHR¹)_(y)OR¹, wherein y is an integer from 1 to 6,

or AY¹ together are

wherein x is an integer from 3 to 5; and

DY² is a halogen, or D is NR¹ and Y² is selected from

cycloalkyl with up to 10 carbon atoms, cycloalkyl with up to 10 carbonatoms substituted with R¹, linear or branched alkyl with up to 10 carbonatoms, CH₂R¹,

wherein x is an integer from 3 to 5,

CH₂CF₃, (CHR¹)_(z)Z¹, wherein z is an integer from 1 to 6, and Z¹ isselected from NR¹ ₂,

wherein x is an integer from 3 to 5,

NY²R¹ together is selected from

wherein Z² is selected from R¹, C(O)R¹, C(O)OR¹, pyridinyl, aryl,

wherein q is an integer from 0 to 6.

Further examples of compounds that show at least this activity andutility are presented in Table 5, where compound activity is also shown.The activity scale used in Table 5 is as follows (numbers areinclusive): “++++” represents between 0 and about 25% of IL6 productioncompared to cells that did not receive compound (or percent of controlIL6 production); “+++” represents between about 25 and about 50% ofcontrol IL6 production; “++” represents between about 50 and about 75%of control IL6 production; and “+” represents between about 75 and 100%of control IL6 production. The note “n.d.” indicates that the activityof the compound was not determined in the given assay. Further note thatany hydrogen atoms that are required for any atom to attain its usualvalence in a structure presented in Table 5, whether a carbon atom or aheteroatom, should be inferred if it is not specifically indicated.

In addition to the above compounds, the compounds shown in Table 7, andcompositions comprising these compounds, also exhibit the activity ofmodulating inflammation activity as measured by the assays taughtherein. The activity scale used in Table 7 is as follows (numbers areinclusive): “+++” represents between about 85 to 100% inhibition of IL6production in the presence of AGE or TNF, as compared to cells that didnot receive any compound; “++” represents between about 65 and about 85%inhibition of IL6 production in the presence of AGE or TNF; and “+”represents between about 50 and about 65% inhibition of IL6 productionin the presence of AGE or TNF. In addition, compounds, to includecompositions thereof, encompassed within the scope of structuresIa-XXIVa, respectively, or listed in Tables 1A and 1E may be likewiseemployed in this embodiment and/or aspect of the present invention. Asbefore, the inclusion of compounds in the categories of the Tablesdisclosed herein are not to be seen as limiting, in that compoundsincluded in such Tables have at least the activity shown for inclusionin the Tables and may have more or other activities. Nor are the Tablesto be seen as limiting in that these are the only compounds disclosedherein that have that activity, representative compounds are shown inthe Tables that have at least that particular activity for inclusion inthe Table. One or more compounds disclosed herein have at least anactivity that has utility in treatment of disease states.

Enhanced formation and accumulation of glycated proteins and AGE arethought to play a major role in the pathogenesis of diabeticcomplications, and atherosclerosis, leading to the development of arange of diabetic complications including nephropathy, retinopathy andneuropathy. There is ample in vivo evidence that suggests thatdiabetes-related complications can be reduced by 1) preventing glycationof proteins, 2) by breaking the cross-links in glycated proteins or 3)by blocking glycated protein interaction with receptors. Despite theimportance of AGE in the pathogenesis of diabetic microangiopathies,there are no currently available medications known to block AGEformation.

Endothelium is the target organ of damage in diabetes. See Laight etal., 15 DIABETES METAB. RES. REV. 274-82 (1999); Stehouwer et al., 34CARDIOVASC. 55-68 (1997). Up-regulation of molecules involved inendothelial inflammation, such as IL-6 and monocyte chemoattractantprotein-1 (MCP-1) leads to endothelial dysfunction and vasculopathy. SeeStehouwer et al., 34 CARDIOVASC. 55-68 (1997); Libby, 247 J. INTERN.MED. 349-58 (2000); Van Lente, 293 CLINICA. CHIMICA. ACTA. 31-52 (2000).

IL-6 is a pro-inflammatory cytokine that is known to play a key role inthe pathogenesis of diabetes and atherosclerosis. See Horii et al., 39KIDNEY INT. SUPPL. 71-5 (1993); Huber et al., 19 ARTERIOSCLER THROMB.VASC. BIOL. 2364-67 (1999); Shikano et al., 85 NEPHRON 81-5 (2000);Pickup et al., 8(67) LIFE SCI. 291-300 (2000). IL-6 also promotes thegrowth of renal mesangial cells thus contributing to nephropathy. SeeKado et al., 36 ACTA. DIABETOL. 67-72 (1999). The serum IL-6 level indiabetic subjects was significantly higher than in normal healthycontrols (3.48+/−3.29 pg/ml vs 0.784+/−0.90 pg/ml, mean+/−SD). Inaddition the urinary IL-6 level is a good indicator of diabeticnephropathy. Serum IL-6 is useful in the evaluation of atherosclerosisand nephropathy.

MCP-1, another pro-inflammatory cytokine is found highly expressed inhuman atherosclerotic lesions and postulated to play a central inmonocyte recruitment into the arterial wall and developing lesions. SeeLibby, 247 J. INTERN. MED. 349-58 (2000). Recent results show that MCP-1is also a key pathogenic molecule in diabetic nephropathy. See Eitner etal., 51 KIDNEY INT. 69-78 (1997); Banba et al. 58 KIDNEY INT. 684-90(2000). Glycated albumin stimulates endothelial production of IL-6 andMCP-1. The effects of glycated albumin on IL-6 production are comparableto that of TNFα, a known inducer of IL-6. These cytokines are known tobe factors in vascular diseases.

The activity of the compounds of the present invention in inhibitingglycated protein- and AGE-induced inflammation can be determined usingthe assays described herein and in U.S. patent application Ser. No.10/026,335, which is herein incorporated in its entirety. Such assayscomprise measurement of the specific activity of biological componentsinvolved in a known cellular response. The assays provide a measurableresponse in which the activity of the compounds is determined. One assaycomprises measurement of the effects of compounds on an inflammatoryresponse by cells to the presence of a stimulating agent. Yet anotherassay comprises endothelial cells that are stimulated by the addition ofa glycated protein, the stimulating agent. The endothelial cells respondby producing specific cytokines. The amount of cytokines produced aredetermined by measurement protocols known to those skilled in the art.The compounds of the present invention are then added to the assay andthe production of cytokines is measured. From the comparison of theassay without the compound with the assay with the compound, thebiological effect of the compound can be determined. The compound mayhave an inhibitory effect, a stimulatory effect, or no effect at all.

The amount and type of cytokine produced can be determined usingimmunological methods, such as ELISA assays. The methods of the presentinvention are not limited by the type of assay used to measure theamount of cytokine produced, and any methods known to those skilled inthe art and later developed can be used to measure the amount ofcytokines produced in response to the stimulating agent and to thecompound having unknown activity.

An aspect of the present invention comprises methods and compositionsfor the treatment of diseases, preconditions or pathologies associatedwith inflammatory cytokines and other inflammation related moleculesincluding, but not limited to IL-6, VCAM-1, AGE-induced MCP-1, (monocytechemoattractant protein 1), heme oxygenase, insulin-like growth factor,selecting, IP-10, MIG and I-TAC, NF-κB, IL-1β (interleukin 1β), IL-11(interleukin 11), m-CSF (macrophage colony stimulating factor),fibrinogen, TNF-α (tumor necrosis factor α), adhesion molecules,selectins, VCAM-1 (Vascular Cell Adhesion Molecule-1), CRP (C-reactiveprotein), and PAI-1 (plasminogen activator inhibitor-1). Examples ofsuch diseases include the pathogenesis of atherosclerosis and thedevelopment of diabetic vasculopathy in type II diabetes. For example,affecting the activity or level of TNFα is a key mediator of tissuedamage following acute or chronic inflammatory reactions. The presentinvention contemplates providing compositions and methods that modulatethe effects of cytokines and inflammatory molecules such as TNFα, IL-6,VCAM-1, IP-10, MIG, I-TAC and AGE-induced MCP-1, and treat theassociated diseases, acute or chronic conditions, preconditions andpathologies.

Assays for determining the activity of compounds capable of modulatinginflammation include those taught in U.S. patent application Ser. Nos.10/026,335 and 09/969,013, which are both expressly incorporated byreference. In general, once the baseline response to the stimulatingagent for the production of cytokines by the endothelial cells isestablished, thus comprising the control levels for the screening assay,the methods comprise addition of compounds of the present invention. Theeffect of the compound on the baseline response is determined bycomparing the amount of cytokine produced in the presence of thestimulating agent and the amount of cytokine produced in the presence ofthe stimulating agent and the compound of the present invention. In apreferred method, compounds that have inhibitory effects on theinflammation of the cells in the presence of glycated albumin are thenused as therapeutic agents. One or more compounds may be added to thescreening assay. Combinations or mixtures of compounds can be added.Different amounts and formulations of the compounds are added todetermine the effects on the screening assay. The screening assay mayalso be used to determine stimulatory compounds or compounds that haveno effects in the assay.

The present invention comprises methods and compositions for thetreatment and prevention of disease, conditions and pathologiesassociated with inflammation. Such methods comprise administration ofcompositions comprising compounds capable of modulating the activity ofmolecules associated with inflammation such as AGE or cytokines or othercellular factors, including release rates or activity, and includecompositions comprising compounds disclosed herein with inflammationmodulating activity. Administration of such compounds that are effectivein modulating inflammation are administered to humans and animalssuspected of having or who have inflammatory diseases, for example,diabetic-induced vasculopathies, autoimmune diseases, renalinsufficiency, Alzheimer's syndrome, and inflammation-induced diseasessuch as atherosclerosis. Effective amounts are administered to suchhumans and animals in dosages that are safe and effective, including,but not limited to, the ranges taught herein. Routes of administrationinclude, but are not limited to, those disclosed herein. As disclosedherein, compositions comprising such compounds may be used inconjunction with other therapeutic agents or in methods comprising stepssuch as altered patient activities, including, but not limited to,changes in exercise or diet.

VI. Cytotoxic Activity

An embodiment of the present invention comprises methods andcompositions comprising compounds that have at least the activity ofcausing cellular death or a cessation of cellular activity, referred toherein as cytotoxic activity. This activity can be used in methods forin vitro or in vivo cytotoxicity. For example, compounds having thisactivity can be selectively delivered to an area within a livingorganism to selectively kill cells in that area. Such methods are usingin treating hyperproliferative cells, such as cancers, or other unwantedcellular growth or cellular activities. One aspect of the inventionprovides compositions comprising compounds that nonselectively killcells. Another aspect of the invention provides compounds thatselectively kill cells, for example, cells that have a particularcellular marker or other identifying characteristic such as metabolicrate or uptake of a particular compound, such as sodium, calcium orthymidine.

The present invention also provides compositions for and methods oftreatment of biological conditions including, but not limited to,conditions for which cytotoxic activity is a treatment. For example, thecompositions and methods for providing compounds that have at least theactivity of cytotoxicity are useful in the treatment or prophylaxis ofat least one hyperproliferative disease in a cell, tissue, organ,animal, or patient including, but not limited to, malignant andnon-malignant cell growth, leukemia, acute leukemia, acute lymphoblasticleukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML),chromic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL),hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma,Hodgkin's disease, a malignamt lymphoma, non-hodgkin's lymphoma,Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectalcarcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignanthistiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy,solid tumors, adenocarcinomas, sarcomas, malignant melanoma, hemangioma,metastatic disease, cancer related bone resorption, cancer related bonepain, and the like.

Compounds of the present invention that have at least the activity ofcytotoxicity are shown in TABLE 4A and B. The compounds shown in thisTable have the activity of cytotoxicty as measured by the assays taughtherein. The inclusion of compounds in the categories of the Tablesdisclosed herein are not to be seen as limiting, in that compoundsincluded in such Tables have at least the activity shown for inclusionin the Table and may have more or other activities. Nor are the Tablesto be seen as limiting in that these are the only compounds disclosedherein that have that activity, representative compounds are shown inthe Tables that have at least that particular activity for inclusion inthe Table. One or more compounds disclosed herein have at least anactivity that has utility in treatment of disease states.

Examples of compounds that show at least this activity and utility areshown in the following formula:

or an ene, a diene, a triene, or an yne derivative thereof; a saturatedderivative thereof; a stereoisomer thereof; or a salt thereof;wherein:

R¹ is in each occurrence independently selected from —H; linear orbranched alkyl with up to 10 carbon atoms; cycloalkyl with up to 10carbon atoms; or aryl;

E is CH or N;

n is an integer from 0 to 3;

X¹ is selected from —H, m-F, m-Cl, m-Br, m-I, m-CN, m-NO₂, M-SO₂R¹, orm-SO₂OR¹, or X¹ and X² together is a fused benzene or pyridine ring;

X² is selected from —H, o-Cl, o-Br, p-OR¹, p-SR¹, p-NR¹ ₂, p-F, p-Cl,p-Br, p-CF₃, p-C(O)OR¹, p-OM, or p-SM, wherein M is selected from Li,Na, K, Mg, or Ca;

A is selected from NR¹ or O, wherein Y¹ is selected from cycloalkyl withup to 10 carbon atoms, linear or branched alkyl with up to 10 carbonatoms, or

when A is NR¹, and wherein Y¹ is selected from R¹ or CH₂R¹ when A is O;or AY¹ is selected from a halogen,

DY² is a halogen, or D is NR¹ and Y² is selected from

(CHR¹)_(x)NR¹ ₂, wherein x is an integer from 1 to 6.

Additional examples of compounds that show at least this activity andutility are shown in the following formula:

or an ene, a diene, a triene, or an yne derivative thereof; a saturatedderivative thereof; a stereoisomer thereof; or a salt thereof;wherein:

R¹ is in each occurrence independently selected from —H; linear orbranched alkyl with up to 10 carbon atoms; or cycloalkyl with up to 10carbon atoms;

X¹ is in each occurrence independently selected from —H, m-F, m-Cl,m-Br, m-I, m-CN, m-NO₂, M-SO₂R¹, or M-SO₂OR¹;

X² is in each occurrence independently selected from o-CH₃, p-OR¹,p-SR¹, p-NR¹ ₂, or p-OM or p-SM, wherein M is selected from Li, Na, K,Mg, or Ca;

Y¹ is selected from cycloalkyl with up to 10 carbon atoms;

wherein n is 1 or 2; or

Y² is selected from

Further examples of compounds that show at least this activity andutility are presented in Tables 4A and 4B. The compound nomenclature ofTables 4A and 4B, as in the other tables presented herein, was generatedusing Autonom, where the name provided may be the Beilstein or CASversion of the chemical name. Note that any hydrogen atoms that arerequired for any atom to attain its usual valence in a structurepresented in Tables 4A and 4B, whether a carbon atom or a heteroatom,should be inferred if it is not specifically indicated. In addition,compounds, to include compositions thereof, encompassed within the scopeof structures Ia-XXIVa, respectively, or listed in Tables 1A and 1E maybe likewise employed in this embodiment and/or aspect of the presentinvention.

Assays for determining the activity of compounds capable of cytotoxicactivity include those taught in herein and others that are well knownin the art. In general, to determine if there is cytotoxic activityassociated with a compound, cells of a particular type, in a growingstage or a quiescient stage, are treated with the compound of interest.Various parameters of cell death or cessation are used to measure theeffects of the compound. For example, the amount of nucleic acid orprotein synthesis can be measured or visual observation of the state ofthe cells, such as release from the substrate, can be used to measurethe state of the cells.

The present invention comprises methods and compositions for thetreatment and prevention of diseases or conditions that present orresult from cellular proliferation or unwanted cellular growth orcellular activity. Such methods comprise administration of compositionscomprising compounds capable of modulating cellular activity or causingcellular death or cessation of growth such as compositions comprisingcompounds disclosed herein that have cytotoxic activity. Administrationof such compounds that are effective in cytotoxic activity areadministered to humans and animals suspected of having or who have, forexample, cancer, overactive tissues such as thyroid or hypothalamus, orcellular conditions where factors are released in unwanted amounts.Effective amounts are administered to such humans and animals in dosagesthat are safe and effective, including, but not limited to, the rangestaught herein. Routes of administration include, but are not limited to,those disclosed herein. As disclosed herein, compositions comprisingsuch compounds may be used in conjunction with other therapeutic agentsor in methods comprising steps such as altered patient activities.

Compound/Composition-Coated Medical Devices

The compounds of the present invention may be used alone or incombination with other agents along with delivery devices to effectivelyprevent and treat the diseases described herein, though particularapplications are found in vascular disease, and in particular, vasculardisease caused by injury and/or by transplantation. Though this examplefocuses on vascular disease, provision of the compounds of the presentinvention with medical devices for treatment of the diseases andconditions capable of being treated with the compounds is contemplatedby the present invention.

Various medical treatment devices utilized in the treatment of vasculardisease may ultimately induce further complications. For example,balloon angioplasty is a procedure utilized to increase blood flowthrough an artery and is the predominant treatment for coronary vesselstenosis. However, the procedure typically causes a certain degree ofdamage to the vessel wall, thereby creating new problems or exacerbatingthe original problem at a point later in time. Although other proceduresand diseases may cause similar injury, exemplary embodiments of thepresent invention will be described with respect to the treatment ofrestenosis and related complications following percutaneous transluminalcoronary angioplasty and other similar arterial/venous procedures,including the joining of arteries, veins and other fluid carryingconduits in other organs or sites of the body, such as the liver, lung,bladder, kidney, brain, prostate, neck and legs.

The local delivery of a compound of the present invention and, in someembodiments, along with other therapeutic agents, from a stent preventsvessel recoil and remodeling through the scaffolding action of thestent. The activity of compound provided, with or without othertherapeutic agents, helps determine for which application, to treatwhich disease, the coated medical device is being administered. Forexample, compound-coated stents can prevent multiple components ofneointimal hyperplasia or restenosis as well as reduce inflammation andthrombosis. Local administration of a compound of the present inventionand other therapeutic agents to stented coronary arteries may also haveadditional therapeutic benefit. For example, higher tissueconcentrations of the compounds of the present invention and othertherapeutic agents may be achieved utilizing local delivery rather thansystemic administration. In addition, reduced systemic toxicity may beachieved utilizing local delivery rather than systemic administrationwhile maintaining higher tissue concentrations. In utilizing localdelivery from a stent rather than systemic administration, a singleprocedure may suffice with better patient compliance. An additionalbenefit of combination therapeutic agent and/or compound therapy may beto reduce the dose of each of the therapeutic agents, thereby limitingtoxicity, while still achieving a reduction in restenosis, inflammationand thrombosis. Local stent-based therapy is therefore a means ofimproving the therapeutic ratio (efficacy/toxicity) of anti-restenosis,anti-inflammatory, and anti-thrombotic therapeutic agents.

Although exemplary embodiments of the invention will be described withrespect to the treatment of restenosis and other related complications,it is important to note that the local delivery of a compound of thepresent invention, alone or as part of a therapeutic agent combination,may be utilized to treat a wide variety of conditions utilizing anynumber of medical devices, or to enhance the function and/or life of thedevice. For example, intraocular lenses, placed to restore vision aftercataract surgery is often compromised by the formation of a secondarycataract. The latter is often a result of cellular overgrowth on thelens surface and can be potentially minimized by combining one or morecompounds of the present invention having activity that is effective inpreventing unwanted cellular growth with the device. Other medicaldevices that often fail due to tissue in-growth or accumulation ofproteinaceous material in, on and around the device, such as shunts forhydrocephalus, dialysis grafts, colostomy bag attachment devices, eardrainage tubes, leads for pace makers and implantable defibrillators canalso benefit from the combinations of the compounds of the presentinvention, possibly other pharmaceutical agents, and the devices. Othersurgical devices, sutures, staples, anastomosis devices, vertebraldisks, bone pins, suture anchors, hemostatic barriers, clamps, screws,plates, clips, vascular implants, tissue adhesives and sealants, tissuescaffolds, various types of dressings, bone substitutes, intraluminaldevices, and vascular supports could also provide enhanced patientbenefit using this compound-device combination approach. Essentially,any type of medical device may be coated in some fashion with at leastone compound of the present invention, alone or as part of a therapeuticagent combination, which enhances treatment over the use of the deviceor therapeutic agent without combination with the compound.

As disclosed supra, the compounds of the present invention can beadministered in combinational therapies with other therapeutic agents,and are not limited to only the other therapeutic agents disclosedherein. Thus, the present invention also contemplates, in addition tovarious medical devices, the coatings on these devices may be used todeliver a compound of the present invention in combination with othertherapeutic agents. This illustrative list of therapeutic agents can beadministered through pharmeutical means or in association with medicaldevices and such therapeutic agents include, but are not limited to,antiproliferative/antimitotic agents including natural products such asvinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine),paclitaxel, epidipodophyllotoxins (e.g., etoposide, teniposide),antibiotics (dactinomycin (actinomycin D) daunorubicin, doxorubicin andidarubicin), anthracyclines, mitoxantrone, bleomycins, plicamycin(mithramycin) and mitomycin, enzymes (L-asparaginase which systemicallymetabolizes L-asparagine and deprives cells which do not have thecapacity to synthesize their own asparagine); antiplatelet agents suchas G(GP) IIb/IIIa inhibitors and vitronectin receptor antagonists;antiproliferative/antimitotic alkylating agents such as nitrogenmustards (mechlorethamine, cyclophosphamide and analogs, melphalan,chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine andthiotepa), alkyl sulfonates-busulfan, nirtosoureas (carmustine (BCNU)and analogs, streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate), pyrimidine analogs (fluorouracil, floxuridine, andcytarabine), purine analogs and related inhibitors (mercaptopurine,thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine));platinum coordination complexes (cisplatin, carboplatin), procarbazine,hydroxyurea, mitotane, aminoglutethimide; hormones (e.g. estrogen);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory; antisecretory (breveldin);anti-inflammatory agents such as adrenocortical steroids (cortisol,cortisone, fludrocortisone, prednisone, prednisolone,6α-methylprednisolone, triamcinolone, betamethasone, and dexamethasone),non-steroidal agents (salicylic acid derivatives, i.e., aspirin;para-aminophenol derivatives, i.e., acetominophen; indole and indeneacetic acids (indomethacin, sulindac, and etodalac), heteroaryl aceticacids (tolmetin, diclofenac, and ketorolac), arylpropionic acids(ibuprofen and derivatives), anthranilic acids (mefenamic acid, andmeclofenamic acid), enolic acids (piroxicam, tenoxicam, phenylbutazone,and oxyphenthatrazone), nabumetone, gold compounds (auranofin,aurothioglucose, gold sodium thiomalate); immunosuppressives.(Cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine,mycophenolate mofetil); angiogenic agents: vascular endothelial growthfactor (VEGF), fibroblast growth factor (FGF); angiotensin receptorblockers; nitric oxide donors; anti-sense oligionucleotides andcombinations thereof; cell cycle inhibitors, mTOR inhibitors, and growthfactor signal transduction kinase inhibitors.

Although any number of stents may be utilized in accordance with thepresent invention, for simplicity, a limited number of stents will bedescribed in exemplary embodiments of the present invention. The skilledartisan will recognize that any number of stents may be utilized inconnection with the present invention. In addition, as stated above,other medical devices may be utilized. For example, though stents aredescribed, sleeves outside the vessels are also contemplated, as areother medical devices that can provide a substrate for administrationfor at least one of the compounds of the present invention.

A stent is commonly used as a tubular structure left inside the lumen ofa duct to relieve an obstruction. Typically, stents are inserted intothe lumen in a non-expanded form and are then expanded autonomously, orwith the aid of a second device in situ. A common method of expansionoccurs through the use of a catheter-mounted, angioplasty balloon thatis inflated within the stenosed vessel or body passageway in order toshear and disrupt the obstructions associated with the wall componentsof the vessel and to obtain an enlarged lumen.

A stent may resemble an expandable cylinder and may comprise afenestrated structure for placement in a blood vessel, duct or lumen tohold the vessel, duct or lumen open, more particularly for protecting asegment of artery from restenosis after angioplasty. The stent may beexpanded circumferentially and maintained in an expanded configurationthat is circumferentially or radially rigid. The stent may be axiallyflexible and when flexed at a band, for example, the stent avoids anyexternally protruding component parts.

The stent may be fabricated utilizing any number of methods. Forexample, the stent may be fabricated from a hollow or formed stainlesssteel tube that may be machined using lasers, electric dischargemilling, chemical etching or other means. The stent is inserted into thebody and placed at the desired site in an unexpanded form. In oneembodiment, expansion may be effected in a blood vessel by a ballooncatheter, where the final diameter of the stent is a function of thediameter of the balloon catheter used. It should be appreciated that astent in accordance with the present invention may be embodied in ashape-memory material including, for example, an appropriate alloy ofnickel and titanium or stainless steel.

Structures formed from stainless steel may be made self-expanding byconfiguring the stainless steel in a predetermined manner, for example,by twisting it into a braided configuration. In this embodiment, afterthe stent has been formed it may be compressed so as to occupy a spacesufficiently small as to permit its insertion in a blood vessel or othertissue by insertion means, wherein the insertion means include asuitable catheter, or flexible rod. Upon emerging from the catheter, thestent may be configured to expand into the desired configuration wherethe expansion is automatic or triggered by a change in pressure,temperature or electrical stimulation.

Furthermore, a stent may be modified to comprise one or more reservoirs.Each of the reservoirs may be opened or closed as desired. Thesereservoirs may be specifically designed to hold the the compound orcompound/therapeutic agent combination to be delivered. Regardless ofthe design of the stent, it is preferable to have the compound orcompound/therapeutic agent combination dosage applied with enoughspecificity and a sufficient concentration to provide an effectivedosage in the affected area. In this regard, the reservoir size in thebands is preferably sized to adequately apply the the compound orcompound/therapeutic agent combination dosage at the desired locationand in the desired amount.

In an alternative embodiment, the entire inner and outer surface of thestent may be coated with the compound or compound/therapeutic agentcombination in therapeutic dosage amounts. The coating techniques mayvary depending on the the compound or compound/therapeutic agentcombination. Also, the coating techniques may vary depending on thematerial comprising the stent or other intraluminal medical device.

One or more compounds of the present invention and, in some instances,other therapeutic agents as a combination, may be incorporated onto oraffixed to the stent in a number of ways. In one embodiment, thecompound is directly incorporated into a polymeric matrix and sprayedonto the outer surface of the stent. The compound elutes from thepolymeric matrix over time and enters the surrounding tissue. Thecompound preferably remains on the stent for at least three days up toapproximately six months, and more preferably between seven and thirtydays.

Any number of non-erodible polymers may be utilized in conjunction withthe compound, and such polyermic compositions are well known in the art.In one embodiment, the polymeric matrix comprises two layers. The baselayer comprises a solution of poly(ethylene-covinylacetate) andpolybutylmethacrylate. The compound is incorporated into this baselayer. The outer layer comprises only polybutylmethacrylate and acts asa diffusion barrier to prevent the compound from eluting too quickly.The thickness of the outer layer or topcoat determines the rate at whichthe compound elutes from the matrix. Essentially, the compound elutesfrom the matrix by diffusion through the polymer matrix. Polymers arepermeable, thereby allowing solids, liquids and gases to escapetherefrom. The total thickness of the polymeric matrix is in the rangefrom about one micron to about twenty microns or greater. It isimportant to note that primer layers and metal surface treatments may beutilized before the polymeric matrix is affixed to the medical device.For example, acid cleaning, alkaline (base) cleaning, salinization andparylene deposition may be used as part of the overall process describedabove.

The poly(ethylene-co-vinylacetate), polybutylmethacrylate and compoundsolution may be incorporated into or onto the stent in a number of ways.For example, the solution may be sprayed onto the stent or the stent maybe dipped into the solution. Other methods include spin coating andplasma polymerization. In one embodiment, the solution is sprayed ontothe stent and then allowed to dry. In another embodiment, the solutionmay be electrically charged to one polarity and the stent electricallycharged to the opposite polarity. In this manner, the solution and stentwill be attracted to one another. In using this type of sprayingprocess, waste may be reduced and more precise control over thethickness of the coat may be achieved.

Drug-coated stents are manufactured by a number of companies includingJohnson & Johnson, Inc. (New Brunswick, N.J.), Guidant Corp. (SantaClara, Calif.), Medtronic, Inc. (Minneapolis, Minn.), Cook GroupIncorporated (Bloomington, Ind.), Abbott Labs., Inc. (Abbott Park,Ill.), and Boston Scientific Corp. (Natick, Mass.). See e.g., U.S. Pat.No. 6,273,913; U.S. Patent Application No. 20020051730; WO 02/26271; andWO 02/26139, each expressly entirely incorporated herein by reference.

Expression Profiles and Microarray Methods of Use

Other aspects of the present invention comprise compositions and methodsfor microarray devices. Such microarray devices and methods comprise avariety of microarrays that may be used, for example, to study andmonitor gene expression in response to treatment with the compounds ofthe present invention. The microarrays may comprise nucleic acidsequences, carbohydrates or proteins that are determinative for specificcells, tissues, species, disease states, prognoses, disease progression,or any other combination of molecules that can be used to determine aneffect of one or more of the compounds of the present invention

For example, the microarrays of the present invention may be derivedfrom, or representative of, for example, a specific organism or celltype, including human microarrays, vascular microarrays, inflammationmicroarrays, cancer microarrays, apoptosis microarrays, oncogene andtumor suppressor microarrays, cell-cell interaction microarrays,cytokine and cytokine receptor microarrays, blood microarrays, cellcycle microarrays, neuroarrays, mouse microarrays, and rat microarrays,or combinations thereof. In further embodiments, the microarrays mayrepresent diseases including cardiovascular diseases, vasculopathicconditions, inflammatory diseases, autoimmune diseases, neurologicaldiseases, immunological diseases, various cancers, infectious diseases,endocrine disorders, and genetic diseases.

Alternatively, the microarrays useful in assessing the efficacy of thecompounds of the present invention may represent a particular tissuetype including, but not limited to, heart, liver, prostate, lung, nerve,muscle, or connective tissue; preferably coronary artery endothelium,umbilical artery endothelium, umbilical vein endothelium, aorticendothelium, dermal microvascular endothelium, pulmonary arteryendothelium, myometrium microvascular endothelium, keratinocyteepithelium, bronchial epithelium, mammary epithelium, prostateepithelium, renal cortical epithelium, renal proximal tubule epithelium,small airway epithelium, renal epithelium, umbilical artery smoothmuscle, neonatal dermal fibroblast, pulmonary artery smooth muscle,dermal fibroblast, neural progenitor cells, skeletal muscle, astrocytes,aortic smooth muscle, mesangial cells, coronary artery smooth muscle,bronchial smooth muscle, uterine smooth muscle, lung fibroblast,osteoblasts, prostate stromal cells, or combinations thereof.

The present invention further contemplates microarrays comprising a geneexpression profile comprising one or more polynucleotide sequencesincluding complementary and homologous sequences, wherein said geneexpression profile is generated from a cell type treated with a compoundof the present invention and is selected from the group comprisingcoronary artery endothelium, umbilical artery endothelium, umbilicalvein endothelium, aortic endothelium, dermal microvascular endothelium,pulmonary artery endothelium, myometrium microvascular endothelium,keratinocyte epithelium, bronchial epithelium, mammary epithelium,prostate epithelium, renal cortical epithelium, renal proximal tubuleepithelium, small airway epithelium, renal epithelium, umbilical arterysmooth muscle, neonatal dermal fibroblast, pulmonary artery smoothmuscle, dermal fibroblast, neural progenitor cells, skeletal muscle,astrocytes, aortic smooth muscle, mesangial cells, coronary arterysmooth muscle, bronchial smooth muscle, uterine smooth muscle, lungfibroblast, osteoblasts, and prostate stromal cells.

The present invention contemplates microarrays comprising one or moreprotein-binding agents, wherein a protein expression profile isgenerated from a cell type treated with a compound of the presentinvention and is selected from the group comprising coronary arteryendothelium, umbilical artery endothelium, umbilical vein endothelium,aortic endothelium, dermal microvascular endothelium, pulmonary arteryendothelium, myometrium microvascular endothelium, keratinocyteepithelium, bronchial epithelium, mammary epithelium, prostateepithelium, renal cortical epithelium, renal proximal tubule epithelium,small airway epithelium, renal epithelium, umbilical artery smoothmuscle, neonatal dermal fibroblast, pulmonary artery smooth muscle,dermal fibroblast, neural progenitor cells, skeletal muscle, astrocytes,aortic smooth muscle, mesangial cells, coronary artery smooth muscle,bronchial smooth muscle, uterine smooth muscle, lung fibroblast,osteoblasts, and prostate stromal cells.

More specifically, the present invention contemplates methods for thereproducible measurement and assessment of the expression of specificmRNAs or proteins in, for example, a specific set of cells. One methodcombines and utilizes the techniques of laser capture microdissection,T7-based RNA amplification, production of cDNA from amplified RNA, andDNA microarrays containing immobilized DNA molecules for a wide varietyof specific genes, including HSPGs such as perlecan, to produce aprofile of gene expression analysis for very small numbers of specificcells. The desired cells are individually identified and attached to asubstrate by the laser capture technique, and the captured cells arethen separated from the remaining cells. RNA is then extracted from thecaptured cells and amplified about one million-fold using the T7-basedamplification technique, and cDNA may be prepared from the amplifiedRNA. A wide variety of specific DNA molecules are prepared thathybridize with specific polynucleotides of the microarray, and the DNAmolecules are immobilized on a suitable substrate. The cDNA made fromthe captured cells is applied to the microarray under conditions thatallow hybridization of the cDNA to the immobilized DNA on themicroarray. The expression profile of the captured cells is obtainedfrom the analysis of the hybridization results using the amplified RNAor cDNA made from the amplified RNA of the captured cells, and thespecific immobilized DNA molecules on the microarray. The hybridizationresults demonstrate, for example, which genes of those represented onthe microarray as probes are hybridized to cDNA from the captured cells,and/or the amount of specific gene expression. The hybridization resultsrepresent the gene expression profile of the captured cells. The geneexpression profile of the captured cells can be used to compare the geneexpression profile of a different set of captured cells. For example,gene expression profiles may be generated from cells treated (and nottreated) with a compound of the present invention. The similarities anddifferences provide useful information for determining the differencesbetween the same cell type under different conditions, morespecifically, the change in gene expression in response to treatmentwith a compound of the present invention.

The techniques used for gene expression analysis are likewise applicablein the context of protein expression profiles. Total protein may beisolated from a cell sample and hybridized to a microarray comprising aplurality of protein-binding agents, which may include antibodies,receptor proteins, small molecules, and the like. Using any of severalassays known in the art, hybridization may be detected and analyzed asdescribed above. In the case of fluorescent detection, algorithms may beused to extract a protein expression profile representative of theparticular cell type. In this regard, the change in protein expressionin response to treatment of cells with a compound of the presentinvention may be evaluated.

Thus, in one aspect, the present invention comprises at least onemicroarray corresponding to a population of genes isolated from aparticular tissue or cell type in methods that is used to detect changesin gene transcription levels that result from exposing the selectedtissue or cells to at least one compound of the present invention. Inthis embodiment, a biological sample derived from an organism, or anestablished cell line, may be exposed to at least one compound of thepresent invention in vivo or ex vivo. Thereafter, the gene transcripts,primarily mRNA, of the tissue or cells are isolated by methodswell-known in the art. SAMBROOK ET AL., MOLECULAR CLONING: A LAB. MANUAL(2001). The isolated transcripts are then contacted with a microarrayunder conditions where the transcripts hybridize with a correspondingprobe to form hybridization pairs. Thus, the microarray provides a modelof the transcriptional responsiveness following exposure to at least onecompound of the present invention. Such information can be used todetermine therapeutic candidates. A hybridization signal may then bedetected at each hybridization pair to obtain a gene expression profile.

Gene and/or protein expression profiles and microarrays may also be usedto identify activating or non-activating compounds of a particular genesuch as perlecan or other HSPG. Compounds that increase transcriptionrates or stimulate, maintain, or stabilize the activity of a protein areconsidered activating, and compounds that decrease rates or inhibit theactivity of a protein are non-activating. Moreover, the biologicaleffects of a compound may be reflected in the biological state of acell. This state is characterized by the cellular constituents. Oneaspect of the biological state of a cell is its transcriptional state.The transcriptional state of a cell includes the identities and amountsof the constituent RNA species, especially mRNAs, in the cell under agiven set of conditions. Thus, the gene expression profiles,microarrays, and algorithms discussed herein may be used to analyze andcharacterize the transcriptional state of a given cell or tissuefollowing exposure to an activating or non-activating compound,specifically, a compound of the present invention.

Microarray techniques and methods for analyzing results are well knownin the art. See U.S. Pat. Nos. 6,263,287; 6,239,209; 6,218,122;6,197,599; 6,156,501; 5,874,219; 5,837,832; 5,700,637; 5,445,934; U.S.Patent Application Nos. 2001/0014461 A1; 2001/0039016 A1; 2001/0034023A1; WO 01/94946; and WO 01/77668. See also, Haab et al., 2 GENOMEBIOLOGY 1-12 (2001); Brown et al., 97 PROC. NATL. ACAD. SCI. USA 262-7(2000); Getz et al., 97 PROC. NATL. ACAD. SCI. USA 12079-84 (2000);Harrington et al., 3 CURRENT OPINION MICROBIOL 285-91 (2000); Holter etal., 97 PROC. NATL. ACAD. SCI. USA 8409-14 (2000); MacBeath et al., 289SCIENCE 1760-63 (2000); Duggan et al., 21 NATURE GENET 10-14 (1999);Lipshutz et al., 21 NATURE GENET 5-9 (1999); Eisen et al., 95 PROC.NATL. ACAD. SCI. USA 14863-68 (1998); Ermolaeva et al., 20 NATURE GENET.19-23 (1998); Hacia et al., 26 NUCLEIC ACIDS RES. 3865-66 (1998);Lockhart et al., NUCLEIC ACIDS SYMP. SER. 11-12 (1998); Schena et al.,16 TRENDS BIOTECHNOL. 301-6 (1998); Shalon, 46 PATHOL. BIOL. 107-9(1998); Welford et al., 26 NUCLEIC ACID RES. 3059-65 (1998); Blanchardet al., 11 BIOSENSORS BIOELECTRONICS 687-90 (1996); Lockhart et al., 14NATURE BIOTECHNOL. 1675-80 (1996); Schena et al., 93 PROC. NATL. ACAD.SCI. USA 10614-19 (1996); Tomayo et al., 96 PROC. NATL. ACAD. SCI. USA2907-12 (1996); Schena et al., 270 SCIENCE 467-70 (1995)

Database Creation, Database Access and Associated Methods of Use

Another embodiment of the present invention comprises a variety ofmethods for managing or using data related to the compounds, methods ofmaking the compounds, methods of using and administering the compounds,and diagnosing, prognosing and following the outcomes associated withdiseases in which the compounds are effective in treating. For example,methods for providing diagnostics and predictors relating tobiomolecules including HSPGS, particularly, perlecan, are contemplatedby the present invention. Also within the scope of this invention aremethods providing diagnostics and predictors relating to the efficacy ofthe compounds of the present invention. The present invention furthercontemplates methods of providing expression profile databases, andmethods for producing such databases, for normal and diseased tissues.

The expression profile database may be an internal database designed toinclude annotation information about the expression profiles generatedto assess the effect of the compounds of the present invention andthrough other sources and methods. Such information may include, forexample, the databases in which a given biomolecule was found, patientinformation associated with the expression profile, including age,cancer or tumor type or progression, information related to a compoundof the present invention such as dosage and administration information,descriptive information about related cDNAs associated with thesequence, tissue or cell source, sequence data obtained from externalsources, expression profiles for a given gene and the related diseasestate or course of disease, for example whether the expression profilerelates to or signifies a particular disease state, and preparationmethods. The expression profiles may be based on protein and/orpolynucleotide microarray data obtained from publicly available orproprietary sources. The database may be divided into two sections: onefor storing the sequences and related expression profiles and the otherfor storing the associated information. This database may be maintainedas a private database with a firewall within the central computerfacility. However, this invention is not so limited and the expressionprofile database may be made available to the public.

The database may be a network system connecting the network server withclients. The network may be any one of a number of conventional networksystems, including a local area network (LAN) or a wide area network(WAN), as is known in the art (e.g., Ethernet). The server may includesoftware to access database information for processing user requests,and to provide an interface for serving information to client machines.The server may support the World Wide Web and maintain a website and Webbrowser for client use. Client/server environments, database servers,and networks are well documented in the technical, trade, and patentliterature.

Through the Web browser, clients may construct search requests forretrieving data from, for example, a microarray database and anexpression profile database. For example, the user may “point and click”to user interface elements such as buttons, pull down menus, and scrollbars. The client requests may be transmitted to a Web application thatformats them to produce a query that may be used to gather informationfrom the system database, based, for example, on microarray orexpression data obtained by the client, and/or other phenotypic orgenotypic information. Specifically, the client may submit expressiondata based on microarray expression profiles obtained from a patienttreated with a compound of the present invention and use the system toobtain a diagnosis based on that information based on a comparison bythe system of the client expression data with the expression datacontained in the database. By way of example, the system compares theexpression profiles submitted by the client with expression profilescontained in the database and then provides the client with diagnosticinformation based on the best match of the client expression profileswith the database profiles. Thus, in one aspect, the comparison ofexpression profiles aids the clinician in determining the effectivenessof treatment with a compound of the present invention. Based on such acomparison, the clinician may alter or adjust the treatment regimen.

In addition, the website may provide hypertext links to public databasessuch as GenBank and associated databases maintained by the NationalCenter for Biotechnology Information (NCBI), part of the NationalLibrary of Medicine as well as, any links providing relevant informationfor gene expression analysis, genetic disorders, scientific literature,and the like. Information including, but not limited to, identifiers,identifier types, biomolecular sequences, common cluster identifiers(GenBank, Unigene, Incyte template identifiers, and so forth) andspecies names associated with each gene, is contemplated.

The present invention also provides a system for accessing and comparingbioinformation, specifically expression profiles and other informationwhich is useful in the context of the compositions and methods of thepresent invention. In one embodiment, the computer system may comprise acomputer processor, suitable memory that is operatively coupled to thecomputer processor, and a computer process stored in the memory thatexecutes in the computer processor and which comprises a means formatching an expression profile of a biomolecular sequence from a patientwith expression profile and sequence identification information ofbiomolecular sequences in a database. More specifically, the computersystem is used to match an expression profile generated from abiological sample treated with a compound of the present invention withexpression profile and other information in a database.

Furthermore, the system for accessing and comparing informationcontained in biomolecular databases comprises a computer programcomprising computer code providing an algorithm for matching anexpression profile generated from a patient, for example, treated with acompound of the present invention, with expression profile and sequenceidentification information of biomolecular sequences in a biomoleculardatabase.

The present invention contemplates, in one embodiment, the use of aGraphical User Interface (“GUI”) for the access of expression profileinformation stored in a biomolecular database. In a specific embodiment,the GUI may be composed of two frames. A first frame may contain aselectable list of biomolecular databases accessible by the user. When abiomolecular database is selected in the first frame, a second frame maydisplay information resulting from the pair-wise comparison of theexpression profile database with the client-supplied expression profileas described above, along with any other phenotypic or genotypicinformation.

The second frame of the GUI may contain a listing of biomolecularsequence expression information and profiles contained in the selecteddatabase. Furthermore, the second frame may allow the user to select asubset, including all of the biomolecular sequences, and to perform anoperation on the list of biomolecular sequences. In one embodiment, theuser may select the subset of biomolecular sequences by selecting aselection box associated with each biomolecular sequence. In anotherembodiment, the operations that may be performed include, but are notlimited to, downloading all listed biomolecular sequences to a databasespreadsheet with classification information, saving the selected subsetof biomolecular sequences to a user file, downloading all listedbiomolecular sequences to a database spreadsheet without classificationinformation, and displaying classification information on a selectedsubset of biomolecular sequences.

If the user chooses to display classification information on a selectedsubset of biomolecular sequences, a second GUI may be presented to theuser. In one embodiment, the second GUI may contain a listing of one ormore external databases used to create the expression profile databasesas described above. Furthermore, for each external database, the GUI maydisplay a list of one or more fields associated with each externaldatabase. In yet another embodiment, the GUI may allow the user toselect or deselect each of the one or more fields displayed in thesecond GUI. In yet another embodiment, the GUI may allow the user toselect or deselect each of the one or more external databases.

The methods of the present application futher relate to the commercialand other uses of the compositions and methodologies of the presentinvention. In one aspect, the methods include the marketing, sale, orlicensing of the compositions and methodologies of the present inventionin the context of providing consumers, i.e., patients, medicalpractitioners, medical service providers, researchers, andpharmaceutical distributors and manufacturers, with expression profiledatabases including, in particular, databases produced in accordancewith the use of the compounds of the present invention.

In another embodiment, the methods of the present invention includeestablishing a distribution system for distributing the pharmaceuticalcompositions of the present invention for sale, and may optionallyinclude establishing a sales group for marketing the pharmaceuticalcomposition. Yet another aspect of the present invention provides amethod of conducting target discovery comprising identifying, by one ormore of the above drug discovery methods, a test compound, as describedabove, which modulates the level of expression of a gene or the activityof a gene product such as perlecan; conducting therapeutic profiling ofagents identified, or further analogs thereof, for efficacy and toxicityin animals; and optionally formulating a pharmaceutical compositionincluding one or more of the agents identified as having an acceptabletherapeutic profile; and optionally licensing or selling, the rights forfurther drug development of said identified agents.

Pharmaceutical Compositions

In addition to the compounds disclosed herein, the pharmaceuticalcompositions of the present invention can further comprise at least oneof any suitable auxiliary such as, but not limited to, diluent, binder,stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvantor the like. Pharmaceutically acceptable auxiliaries are preferred.Examples and methods of preparing such sterile solutions are well knownin the art and can be found in well known texts such as, but not limitedto, REMINGTON'S PHARMACEUTICAL SCIENCES (Gennaro, Ed., 18th Edition,Mack Publishing Co. (1990)). Pharmaceutically acceptable carriers can beroutinely selected that are suitable for the mode of administration,solubility and/or stability of the compound.

Pharmaceutical excipients and additives useful in the present inventioninclude, but are not limited to, proteins, peptides, amino acids,lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-,tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols,aldonic acids, esterified sugars and the like; and polysaccharides orsugar polymers), which can be present singly or in combination,comprising alone or in combination in ranges of 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid components, which can alsofunction in a buffering capacity, include alanine, glycine, arginine,betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine,leucine, isoleucine, valine, methionine, phenylalanine, aspartame, andthe like.

Carbohydrate excipients suitable for use in the present inventioninclude, for example, monosaccharides such as fructose, maltose,galactose, glucose, D-mannose, sorbose, and the like; disaccharides,such as lactose, sucrose, trehalose, cellobiose, and the like;polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans,starches, and the like; and alditols, such as mannitol, xylitol,maltitol, lactitol, xylitol, sorbitol (glucitol), myoinositol and thelike.

The pharmaceutical compositions comprising the compounds of the presentinvention can also include a buffer or a pH adjusting agent. Typically,the buffer is a salt prepared from an organic acid or base.Representative buffers include organic acid salts such as salts ofcitric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid,succinic acid, acetic acid, or phthalic acid; Tris, tromethaminehydrochloride, or phosphate buffers.

Additionally, pharmaceutical compositions of the invention can includepolymeric excipients/additives such as polyvinylpyrrolidones, ficolls (apolymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents,anti-microbial agents, sweeteners, antioxidants, anti-static agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”),lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol),and chelating agents (e.g., EDTA). These and additional knownpharmaceutical excipients and/or additives suitable for use in thepresent invention are known in the art, e.g., as listed in REMINGTON:THE SCIENCE & PRACTICE OF PHARMACY (19^(th) ed., Williams & Williams(1995)) and PHYSICIAN'S DESK REFERENCE (52^(nd) ed., Medical Economics(1998)), the disclosures of which are expressly entirely incorporatedherein by reference.

Pharmaceutical Compositions for Oral Administration

For oral administration in the form of a tablet or capsule, a compoundmay be combined with an oral, non-toxic pharmaceutically acceptableinert carrier such as ethanol, glycerol, water and the like. Moreover,when desired or necessary, suitable binders, lubricants, disintegratingagents, and coloring agents may also be incorporated into the mixture.Suitable binders include, without limitation, starch; gelatin; naturalsugars such as glucose or beta-lactose; corn sweeteners; natural andsynthetic gums such as acacia, tragacanth, or sodium alginate,carboxymethylcellulose; polyethylene glycol; waxes and the like.Lubricants used in these dosage forms include, without limitation,sodium oleate, sodium stearate, magnesium stearate, sodium benzoate,sodium acetate, sodium chloride and the like. Disintegrators include,without limitation, starch, methyl cellulose, agar, bentonite, xanthangum and the like.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil emulsion and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface active ordispersing agent. Molded tablets may be made by molding, in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may be optionally coated or scored and maybe formulated so as to provide a slow or controlled release of theactive ingredient therein.

In addition, the combinations may be incorporated into biodegradablepolymers allowing for sustained release of the compound, the polymersbeing implanted in the vicinity of where drug delivery is desired, forexample, at the site of restenosis. The biodegradable polymers and theiruses are described, for example, in detail in Brem et al., 74 J.NEUROSURG. 441-46 (1991). Suitable examples of sustained-releasecompositions include semipermeable matrices of solid hydrophobicpolymers containing a compound of the present invention, which matricesare in the form of shaped articles, e.g., films, or microcapsules.Examples of sustained-release matrices include polyesters, hydrogels(for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT® (Tap Pharmaceuticals, Inc., Chicago, Ill.) (injectablemicrospheres composed of lactic acid glycolic acid copolymer andleuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

Pharmaceutical Compositions for Parenteral Administration

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes that render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

For parenteral administration, sterile suspensions and solutions aredesired. Isotonic preparations which generally contain suitablepreservatives are employed when intravenous administration is desired.The pharmaceutical compositions may be administered parenterally viainjection of a formulation consisting of the active ingredient dissolvedin an inert liquid carrier. The term “parenteral,” as used herein,includes, but is not limited to, subcutaneous injections, intravenous,intramuscular, intraperitoneal injections, or infusion techniques.Acceptable liquid carriers include, for example, vegetable oils such aspeanut oil, cotton seed oil, sesame oil and the like, as well as organicsolvents such as solketal, glycerol formal and the like. Theformulations may be prepared by dissolving or suspending the activeingredient in the liquid carrier such that the final formulationcontains from about 0.005% to 30% by weight of the active ingredient,i.e., a compound of the present invention.

Pharmaceutical Compositions for Other Routes of Administration

Formulations suitable for topical administration in the mouth includelozenges comprising the ingredients in a flavored basis, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the compound to be administered in a suitableliquid carrier. The liquid forms may include suitably flavoredsuspending or dispersing agents such as the synthetic and natural gums,for example, tragacanth, acacia, methyl-cellulose and the like.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tamports, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The compounds may also be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.REMINGTON'S PHARMACEUTICAL SCIENCES (A. Osol ed., 16th ed. (1980)).

In a specific embodiment, the compounds disclosed herein are formulatedas liposomes. Liposomes containing a compound of the present inventionare prepared by methods known in the art. See, e.g., U.S. Pat. Nos.5,013,556; 4,485,045; 4,544,545; WO 97/38731; Epstein et al., 82 PROC.NATL. ACAD. SCI. USA 3688 (1985); and Hwang et al., 77 PROC. NATL. ACAD.SCI. USA 4030 (1980). The compounds of the present invention can also beadministered in the form of liposome delivery systems such as smallunilamellar vesicles, large unilamellar vesicles, and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids suchas cholesterol, stearylamine or phophatidylcholines.

Compounds of the present invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The compounds of the present invention may alsobe coupled with soluble polymers as targetable drug carriers. Suchpolymers can include polyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamidephenol,polyhydroxyethylaspartamidephenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residue.

Pharmaceutically Acceptable Preservatives

The present invention provides stable formulations as well as preservedsolutions and formulations containing a preservative as well asmulti-use preserved formulations suitable for pharmaceutical orveterinary use, comprising at least one compound disclosed herein in apharmaceutically acceptable formulation. Formulations in accordance withthe present invention may optionally contain at least one knownpreservative. Preservatives include, but are not limited to, phenol,m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol,magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl,propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof inan aqueous diluent. Any suitable concentration or mixture can be used asknown in the art, such as 0.001-5%, or any range or value therein.Non-limiting examples include, no preservative, 0.1-2% m-cresol, 0.1-3%benzyl alcohol, 0.001-0.5% thimerosal, 0.001-2.0% pheno, 0.0005-1.0%alkylparaben(s), and the like.

Other excipients, e.g., isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally added to the diluent. Anisotonicity agent such as glycerin, is commonly used at knownconcentrations. A physiologically tolerated buffer is preferably addedto provide improved pH control. The formulations can cover a wide rangeof pHs, such as from about pH 4 to about pH 10, specifically, a rangefrom about pH 5 to about pH 9, and more specifically, a range of about6.0 to about 8.0. In one aspect, the formulations of the presentinvention have pH between about 6.8 and about 7.8. Suitable buffersinclude phosphate buffers, for example, sodium phosphate and phosphatebuffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators such asEDTA and EGTA can optionally be added to the pharmaceutical compositionsto reduce aggregation. These additives are particularly useful if a pumpor plastic container is used to administer the pharmacueticalcomposition. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the composition to aggregate.

During any of the processes for preparation of the compounds of thepresent invention, it may be necessary and/or desirable to protectsensitive or reactive groups on any of the molecules concerned. This maybe achieved by means of conventional protecting groups, such as thosedescribed in PROTECTIVE GROUPS IN ORGANIC CHEMISTRY (1973); and GREENEAND WUTS, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (1991). The protectinggroups may be removed at a convenient subsequent stage using methodsknown from the art.

Routes of Administration

The invention further relates to the administration of at least onecompound disclosed herein by the following routes, including, but notlimited to oral, parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, iontophoretic means, ortransdermal means.

Pulmonary/Nasal Administration

There are a several desirable features of an inhalation device foradministering a compound of the present invention. For example, deliveryby the inhalation device is reliable, reproducible, and accurate. Forpulmonary administration, at least one pharmaceutical composition isdelivered in a particle size effective for reaching the lower airways ofthe lung or sinuses. The inhalation device can optionally deliver smalldry particles, e.g. less than about 10 μm, preferably about 1-5 μm, forgood respirability.

According to the invention, at least one pharmaceutical composition canbe delivered by any of a variety of inhalation or nasal devices known inthe art for administration of a therapeutic agent by inhalation. Devicescapable of depositing aerosolized formulations in the sinus cavity oralveoli of a patient include metered dose inhalers, nebulizers, drypowder generators, sprayers, and the like. Other devices suitable fordirecting pulmonary or nasal administration are also known in the art.

All such devices can be used for the administration of a pharmaceuticalcomposition in an aerosol. Such aerosols may comprise either solutions(both aqueous and non aqueous) or solid particles. Metered dose inhalerslike the Ventolin® metered dose inhaler, typically use a propellent gasand require actuation during inspiration. See, e.g., WO 98/35888; WO94/16970. Dry powder inhalers like Turbuhaler® (Astra), Rotahaler®(Glaxo), Diskus® (Glaxo), Spiros® inhaler (Dura), devices marketed byInhale Therapeutics, and the Spinhaler® powder inhaler (Fisons), usebreath-actuation of a mixed powder. See U.S. Pat. Nos. 5,458,135;4,668,218; WO 97/25086; WO 94/08552; WO 94/06498; and EP 0 237 507, eachentirely expressly incorporated herein by reference. Nebulizers likeAERx®, Aradigm, the Ultravent® nebulizer (Mallinckrodt), and the AcornII® nebulizer (Marquest Medical Products), the above references entirelyexpressly incorporated herein by reference, produce aerosols fromsolutions, while metered dose inhalers, dry powder inhalers, etc.generate small particle aerosols. These specific examples ofcommercially available inhalation devices are intended to be arepresentative of specific devices suitable for the practice of theinvention, and are not intended as limiting the scope of the invention.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is administered, i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose.Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

A spray comprising a pharmaceutical composition of the present inventioncan be produced by forcing a suspension or solution of a compounddisclosed herein through a nozzle under pressure. The nozzle size andconfiguration, the applied pressure, and the liquid feed rate can bechosen to achieve the desired output and particle size. An electrospraycan be produced, for example, by an electric field in connection with acapillary or nozzle feed. Advantageously, particles of at least onecompound delivered by a sprayer have a particle size in a range of aboutless than 1 μm to less than about 20 μm.

Pharmaceutical compositions of at least one of the compounds of thepresent invention suitable for use with a sprayer typically include acompound disclosed herein in an aqueous solution at a concentration ofabout 0.1 mg to about 100 mg of a compound disclosed herein per ml ofsolution or mg/gm, or any range or value therein, including, but notlimited to, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,0.2., 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml or mg/gm. Thepharmaceutical composition can include agents such as an excipient, abuffer, an isotonicity agent, a preservative, a surfactant, or otherknown agents of pharmaceutical compositions.

A pharmaceutical composition of the present invention can beadministered by a nebulizer such as a jet nebulizer or an ultrasonicnebulizer. Typically, in a jet nebulizer, a compressed air source isused to create a high-velocity air jet through an orifice. As the gasexpands beyond the nozzle, a low-pressure region is created, which drawsa solution of composition protein through a capillary tube connected toa liquid reservoir. The liquid stream from the capillary tube is shearedinto unstable filaments and droplets as it exits the tube, creating theaerosol. A range of configurations, flow rates, and baffle types can beemployed to achieve the desired performance characteristics from a givenjet nebulizer. In an ultrasonic nebulizer, high-frequency electricalenergy is used to create vibrational, mechanical energy, typicallyemploying a piezoelectric transducer. This energy is transmitted to theformulation of composition protein either directly or through a couplingfluid, creating an aerosol including the composition protein.Advantageously, particles of the pharmaceutical composition delivered bya nebulizer have a particle size range of from about less than 1 μm toless than about 20 μm.

Pharmaceutical compositions comprising a compound of the presentinvention suitable for use with a nebulizer, either jet or ultrasonic,typically include a concentration of about 0.1 mg to about 100 mg of acompound disclosed herein per ml of solution or mg/gm, or any range orvalue therein including, but not limited to, the individual amountsdisclosed for spray compositions. The pharmaceutical composition caninclude other pharmaceutical agents such as an excipient, a buffer, anisotonicity agent, a preservative, a surfactant, and those known in theart for use in nebulizer administration.

In a metered dose inhaler (MDI), a propellant, a compound of the presentinvention, and any excipients or other additives are contained in acannister as a mixture including a liquefied, compressed gas. Actuationof the metering valve releases the mixture as an aerosol, preferablycontaining a particle size range of from about less than 1 μm to lessthan about 20 μm.

The desired aerosol particle size can be obtained by employing aformulation of a compound of the present invention produced by variousmethods known to those of skill in the art including, but not limitedto, jet-milling, spray drying, critical point condensation, and thelike. Suitable metered dose inhalers include those manufactured by 3M orGlaxo and employing a hydrofluorocarbon propellant.

Pharmaceutical compositions for use with a metered-dose inhaler devicewill generally include a finely divided powder containing a compounddisclosed herein as a suspension in a non-aqueous medium, for example,suspended in a propellant with the aid of a surfactant. The propellantcan be any conventional material employed for this purpose such aschlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like. Inone embodiment, the propellant is a hydrofluorocarbon. The surfactantcan be chosen to stabilize the compound of the present invention as asuspension in the propellant, to protect the active agent againstchemical degradation, and the like. Suitable surfactants includesorbitan trioleate, soya lecithin, oleic acid, or the like. In somecases solution aerosols are preferred using solvents such as ethanol.One of ordinary skill in the art will recognize that the methods of thepresent invention can be achieved by pulmonary administration of acompound disclosed herein via devices not described herein.

For absorption through mucosal surfaces, the compositions and methods ofthe present invention for administering a compound disclosed hereininclude an emulsion comprising a plurality of submicron particles, amucoadhesive macromolecule, a bioactive peptide, and an aqueouscontinuous phase, which promotes absorption through mucosal surfaces byachieving mucoadhesion of the emulsion particles. See, e.g., U.S. Pat.No. 5,514,670. Mucous surfaces suitable for application of the emulsionsof the present invention can include corneal, conjunctival, buccal,sublingual, nasal, vaginal, pulmonary, abdominal, intestinal, and rectalroutes of administration. Pharmaceutical compositions for vaginal orrectal administration such as suppositories, can contain as excipients,for example, polyalkyleneglycols, vaseline, cocoa butter, and the like.Pharmaceutical composition s for intranasal administration can be solidand contain excipients, for example, lactose or can be aqueous or oilysolutions of nasal drops. For buccal administration, excipients includesugars, calcium stearate, magnesium stearate, pregelinatined starch, andthe like. See, e.g., U.S. Pat. No. 5,849,695.

In another embodiment, the pharmaceutical compositions of the presentinvention may be administered via transdermal routes using forms oftransdermal skin patches well known to those of ordinary skill in thatart. For transdermal administration, a compound of the present inventionis encapsulated in a delivery device such as a liposome or polymericnanoparticles, microparticle, microcapsule, or microspheres (referred tocollectively as microparticles unless otherwise stated). A number ofsuitable devices are known, including microparticles made of syntheticpolymers such as polyhydroxy acids such as polylactic acid, polyglycolicacid and copolymers thereof, polyorthoesters, polyanhydrides, andpolyphosphazenes, and natural polymers such as collagen, polyaminoacids, albumin and other proteins, alginate and other polysaccharides,and combinations thereof. See, e.g., U.S. Pat. No. 5,814,599. To beadministered in the form of a transdermal delivery system, the dosageadministration may be, for example, continuous rather than intermittentthroughout the dosage regimen.

Formulations suitable for topical administration to the skin may bepresented as ointments, creams, gels and pastes comprising theingredient to be administered in a pharmaceutical acceptable carrier. Apreferred topical delivery system is a transdermal patch comprising acompound of the present invention.

Topical compositions containing a compound of the present invention maybe admixed with a variety of carrier materials well known in the artincluding alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, PPG2 myristyl propionate and the like to form, forexample, alcoholic solutions, topical cleansers, cleansing creams, skingels, skin lotions, and shampoos in cream or gel formulations. Examplesof such carriers and methods of formulation may be found in REMINGTON'SPHARMACEUTICAL SCIENCES (1990). Pharmaceutical formulations may containfrom about 0.005% to about 10% by weight of the active ingredient. Inone embodiment, the pharmaceutical formulations contain from about 0.01%to 5% by weight of the compound of the present invention.

It can be sometimes desirable to deliver the compounds of the presentinvention to the subject over prolonged periods of time, for example,for periods of one week to one year from a single administration.Certain medical devices may be employed to provide a continuousintermittent or on demand dosing of a patient. The devices may be a pumpof diffusion apparatus, or other device containing a reservoir of drugand optionally diagnostic or monitoring components to regulate thedelivery of the drug. Various slow-release, depot or implant dosageforms can be utilized. For example, a dosage form can contain apharmaceutically acceptable non-toxic salt of compound disclosed hereinthat has a low degree of solubility in body fluids, for example, (a) anacid addition salt with a polybasic acid such as phosphoric acid,sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid,alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids,polygalacturonic acid, and the like; (b) a salt with a polyvalent metalcation such as zinc, calcium, bismuth, barium, magnesium, aluminum,copper, cobalt, nickel, cadmium and the like, or with an organic cationformed from e.g., N,N′-dibenzyl-ethylenediamine or ethylenediamine; or(c) combinations of (a) and (b) e.g., a zinc tannate salt. Additionally,the compounds of the present invention or, preferably, a relativelyinsoluble salt such as those just described, can be formulated in a gel,for example, an aluminum monostearate gel with, e.g., sesame oil,suitable for injection. Exemplary salts include, but are not limited to,zinc salts, zinc tannate salts, pamoate salts, and the like. Anothertype of slow-release depot formulation for injection would contain thecompound or salt dispersed or encapsulated in a slow degrading,non-toxic, non-antigenic polymer such as a polylactic acid/polyglycolicacid polymer, for example, as described in U.S. Pat. No. 3,773,919. Thecompounds or relatively insoluble salts thereof such as those describedabove can also be formulated in cholesterol matrix silastic pellets,particularly for use in animals. Additional slow-release, depot orimplant formulations, e.g., gas or liquid liposomes are known in theliterature. See, e.g., U.S. Pat. No. 5,770,222; SUSTAINED AND CONTROLLEDRELEASE DRUG DELIVERY SYSTEMS (1978).

Other examples include provision of the compounds of the presentinvention to be administered by sustained release delivery systemcontaining a biodegradable composition. The biodegradable compositionmay be composed of a biodegradable, water-coagulable, non-polymericmaterial and a biocompatible, non-toxic organic solvent that is miscibleto dispersible in an aqueous medium. The delivery system may beimplanted at an implant site causing the solvent to dissipate, disperseor leach from the composition into surrounding tissue fluid through aresulting microporous matrix.

As used herein, the term “implant site” is meant to include a site, inor on which the non-polymeric composition is applied. Implantation orimplant site can also include the incorporation of the pharmaceuticalcomposition comprising at least one compound of the present inventionwith a solid device. For example, the pharmaceutical composition isincorporated into a coating on a stent that is implanted into a subject.Additionally, other solid or biodegradeable materials can be used as asubstrate on which the pharmaceutical composition is applied. The coatedmaterial, comprising the pharmaceutical composition is then implanted,inserted or is adjacent to the subject or patient. The term“biodegradable” means that the non-polymeric material and/or matrix ofthe implant will degrade over time by the action of enzymes, by simpleor enzymatically catalyzed hydrolytic action and/or by other similarmechanisms in the human body. By “bioerodible,” it is meant that theimplant matrix will erode or degrade over time due, at least in part, tocontact with substances found in the surrounding tissue fluids, cellularaction, and the like. By “bioabsorbable,” it is meant that thenon-polymeric matrix will be broken down and absorbed within the humanbody, for example, by a cell, a tissue, and the like.

Non-polymeric materials that can be used in the composition generallyare those that are biocompatible, substantially insoluble in water andbody fluids, and biodegradable and/or bioerodible. The non-polymericmaterial is capable of being at least partially solubilized in awater-soluble organic solvent. The non-polymeric materials are alsocapable of coagulating or solidifying to form a solid implant matrix.The non-polymeric material is combined with a compatible and suitableorganic solvent to form a composition that has the desired consistencyranging from watery to viscous to a spreadable putty or paste.

Suitable organic solvents are those that are biocompatible,pharmaceutically-acceptable, and will at least partially dissolve thenon-polymeric material. The organic solvent has a solubility in waterranging from miscible to dispersible. Optionally, a pore-forming agentcan be included in the composition to generate additional pores in theimplant matrix. The pore-forming agent can be any organic or inorganic,pharmaceutically-acceptable substance that is substantially soluble inwater or body fluid, and will dissipate from the coagulatingnon-polymeric material and/or the solid matrix of the implant intosurrounding body fluid at the implant site.

The compounds of the present invention are capable of providing a localor systemic biological, physiological or therapeutic effect in the bodyof an animal. In formulating some pharmaceutical compositions describedherein, the compound is preferably soluble or dispersible in thenon-polymeric composition to form a homogeneous mixture, and uponimplantation, becomes incorporated into the implant matrix. As the solidmatrix degrades over time, the compound is capable of being releasedfrom the matrix into the adjacent tissue fluid, and to the pertinentbody tissue or organ, either adjacent to or distant from the implantsite, preferably at a controlled rate. The release of the compound fromthe matrix may be varied, for example, by the solubility of the compoundin an aqueous medium, the distribution of the compound within thematrix, the size, shape, porosity, and solubility and biodegradabilityof the solid matrix. See e.g. U.S. Pat. No. 5,888,533. The amounts andconcentrations of ingredients in the composition administered to thepatient will generally be effective to accomplish the task intended.

Compounds of the present invention may be administered by bioactiveagent delivery systems containing microparticles suspended in a polymermatrix. The microparticles may be microcapsules, microspheres ornanospheres currently known in the art. The microparticles should becapable of being entrained intact within a polymer that is or becomes agel once inside a biological environment. The microparticles can bebiodegradable or non-biodegradable. Many microencapsulation techniquesused to incorporate a bioactive agent into a microparticle carrier aretaught in the art. See e.g. U.S. Pat. Nos. 4,652,441; 5,100,669;4,438,253; and 5,665,428.

A preferred polymeric matrix will be biodegradable and exhibit watersolubility at low temperature and will undergo reversible thermalgelation at physiological mammalian body temperatures. The polymericmatrix is capable of releasing the substance entrained within its matrixover time and in a controlled manner. The polymers are graduallydegraded by enzymatic or non-enzymatic hydrolysis in aqueous orphysiological environments. See e.g. U.S. Pat. No. 6,287,588.

Compounds of the present invention may be administered by a drugdelivery composition comprising microparticles containing at least onechemotherapeutic agent and at least one chemosensitizer suspended in apolymer matrix. The microparticles may be microcapsules, microspheres ornanospheres currently known in the art. The microparticles should bebiodegradable and stable in physiological environments. Themicroparticles also permit diffusion of the chemotherapeutic agent andchemosensitizer from the core through the matrix at a predeterminedrelease rate. Ionic chemotherapeutic agents are suitable for use in thedelivery composition of the invention. Ionic chemosensitizers aresuitable for use in the delivery composition of the invention. The drugdelivery compositions may be delivered to a target site through avariety of known routes of administration. Dosages of thechemotherapeutic agent and chemosensitiThe drug delivery compositionsmay be delivered to a target site through a variety of known routes ofadministration. Dosages of the chemotherapeutic agent andchemosensitizer incorporated in the drug delivery composition willdepend on individual needs, the desired effect and on the chosen routeof administration. See e.g. WO 98/50018.

Dosage Determinations

In general, the compounds disclosed herein may be used alone or inconcert with other therapeutic agents at appropriate dosages defined byroutine testing in order to obtain optimal efficacy while minimizing anypotential toxicity. The dosage regimen utilizing a compound of thepresent invention may be selected in accordance with a variety offactors including type, species, age, weight, sex, medical condition ofthe patient; the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound employed. A physician or veterinarian of ordinaryskill can readily determine and prescribe the effective amount of thedrug required to prevent, counter, or arrest the progress of thecondition.

Optimal precision in achieving concentrations of drug within the rangethat yields maximum efficacy with minimal toxicity may require a regimenbased on the kinetics of the compound's availability to one or moretarget sites. Distribution, equilibrium, and elimination of a drug maybe considered when determining the optimal concentration for a treatmentregimen. The dosages of a compound disclosed herein may be adjusted whencombined to achieve desired effects. On the other hand, dosages of thesevarious therapeutic agents may be independently optimized and combinedto achieve a synergistic result wherein the pathology is reduced morethan it would be if either agent were used alone.

In particular, toxicity and therapeutic efficacy of a compound disclosedherein may be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index and it maybe expressed as the ratio LD₅₀/ED₅₀. Compounds exhibiting largetherapeutic indices are preferred except when cytotoxicity of thecompound is the activity or therapeutic outcome that is desired.Although compounds that exhibit toxic side effects may be used, adelivery system can target such compounds to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects. Generally, the compounds of the present inventionmay be administered in a manner that maximizes efficacy and minimizestoxicity.

Data obtained from cell culture assays and animal studies may be used informulating a range of dosages for use in humans. The dosages of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any compound used in the methods of theinvention, the therapeutically effective dose may be estimated initiallyfrom cell culture assays. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC₅₀(the concentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationmay be used to accurately determine useful doses in humans. Levels inplasma may be measured, for example, by high performance liquidchromatography.

Moreover, the dosage administration of the pharmaceutical compositionsof the present invention may be optimized using apharmacokinetic/pharmacodynamic modeling system. For example, one ormore dosage regimens may be chosen and a pharmacokinetic/pharmacodynamicmodel may be used to determine the pharmacokinetic/pharmacodynamicprofile of one or more dosage regimens. Next, one of the dosage regimensfor administration may be selected which achieves the desiredpharmacokinetic/pharmacodynamic response based on the particularpharmacokinetic/pharmacodynamic profile. See WO 00/67776, which isentirely expressly incorporated herein by reference.

Methods are known in the art for determining effective doses fortherapeutic and prophylactic purposes for the disclosed pharmaceuticalcompositions or the disclosed drug combinations, whether or notformulated in the same composition. For therapeutic purposes, the term“jointly effective amount,” as used herein, means that amount of eachactive compound or pharmaceutical agent, alone or in combination, thatelicits the biological or medicinal response in a tissue system, animalor human that is being sought by a researcher, veterinarian, medicaldoctor or other clinician, which includes alleviation of the symptoms ofthe disease or disorder being treated. For prophylactic purposes (i.e.,inhibiting the onset or progression of a disorder), the term “jointlyeffective amount” refers to that amount of each active compound orpharmaceutical agent, alone or in combination, that inhibits in asubject the onset or progression of a disorder as being sought by aresearcher, veterinarian, medical doctor or other clinician. Thus, thepresent invention provides combinations of two or more therapeuticagents wherein, for example, (a) each therapeutic agent is administeredin an independently therapeutically or prophylactically effectiveamount; (b) at least one therapeutic agent in the combination isadministered in an amount that is sub-therapeutic or subprophylactic ifadministered alone, but is therapeutic or prophylactic when administeredin combination with the second or additional therapeutic agentsaccording to the invention; or (c) both therapeutic agents areadministered in an amount that is subtherapeutic or sub-prophylactic ifadministered alone, but are therapeutic or prophylactic whenadministered together. Combinations of three or more therapeutic agentsare analogously possible. Methods of combination therapy includecoadministration of a single formulation containing all active agents;essentially contemporaneous administration of more than one formulation;and administration of two or more active agents separately formulated.

Dosages

More specifically, the pharmaceutical compositions may be administeredin a single daily dose, or the total daily dosage may be administered individed doses of two, three, or four times daily. In the case of oraladministration, the daily dosage of the compositions may be varied overa wide range from about 0.0001 to about 1,000 mg per patient, per day.The range may more particularly be from about 0.001 mg/kg to 10 mg/kg ofbody weight per day, about 0.1-100 mg, about 1.0-50 mg or about 1.0-20mg per day for adults (at about 60 kg).

The daily dosage of the pharmaceutical compositions may be varied over awide range from about 0.01 to about 1000 mg per adult human per day. Fororal administration, the pharmaceutical compositions are preferablyprovided in the form of tablets containing from about 0.1 mg to about1000 mg of the compound or 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800,900, or 1000 milligrams of the active compound for the symptomaticadjustment of the dosage to the patient to be treated. An effectiveamount of the drug is ordinarily supplied at a dosage level of fromabout 0.1 mg/kg to about 20 mg/kg of body weight per day. In oneembodiment, the range is from about 0.2 mg/kg to about 10 mg/kg of bodyweight per day. In another embodiment, the range is from about 0.5 mg/kgto about 10 mg/kg of body weight per day. The compounds may beadministered on a regimen of about 1 to about 10 times per day.

In the case of injections, it is usually convenient to give by anintravenous route in an amount of about 0.01-30 mg, about 0.1-20 mg orabout 0.1-10 mg per day to adults (at about 60 kg). In the case of otheranimals, the dose calculated for 60 kg may be administered as well.

Doses of a compound of the present invention can optionally include0.0001 to 1,000 mg/kg/administration, or 0.001 to 100.0mg/kg/administration, from 0.01 to 10 mg/kg/administration, from 0.1 to10 mg/kg/administration, including 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500 mg/kg/administration orany range, value or fraction thereof, or to achieve a serumconcentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2.9,3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5,7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20,12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5., 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5, 15, 15.5, 15.9,16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20,20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, and/or 5000 μg/mlserum concentration per single or multiple administration or any range,value or fraction thereof.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of a compound of the presentinvention 0.1 to 100 mg/kg such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, perday, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively or additionally,at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,or 52, or alternatively or additionally, at least one of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years, or anycombination thereof, using single, infusion or repeated doses.

Specifically, the pharmaceutical compositions of the present inventionmay be administered at least once a week over the course of severalweeks. In one embodiment, the pharmaceutical compositions areadministered at least once a week over several weeks to several months.In another embodiment, the pharmaceutical compositions are administeredonce a week over four to eight weeks. In yet another embodiment, thepharmaceutical compositions are administered once a week over fourweeks.

More specifically, the pharmaceutical compositions may be administeredat least once a day for about 2 days, at least once a day for about 3days, at least once a day for about 4 days, at least once a day forabout 5 days, at least once a day for about 6 days, at least once a dayfor about 7 days, at least once a day for about 8 days, at least once aday for about 9 days, at least once a day for about 10 days, at leastonce a day for about 11 days, at least once a day for about 12 days, atleast once a day for about 13 days, at least once a day for about 14days, at least once a day for about 15 days, at least once a day forabout 16 days, at least once a day for about 17 days, at least once aday for about 18 days, at least once a day for about 19 days, at leastonce a day for about 20 days, at least once a day for about 21 days, atleast once a day for about 22 days, at least once a day for about 23days, at least once a day for about 24 days, at least once a day forabout 25 days, at least once a day for about 26 days, at least once aday for about 27 days, at least once a day for about 28 days, at leastonce a day for about 29 days, at least once a day for about 30 days, orat least once a day for about 31 days.

Alternatively, the pharmaceutical compositions may be administered aboutonce every day, about once every 2 days, about once every 3 days, aboutonce every 4 days, about once every 5 days, about once every 6 days,about once every 7 days, about once every 8 days, about once every 9days, about once every 10 days, about once every 11 days, about onceevery 12 days, about once every 13 days, about once every 14 days, aboutonce every 15 days, about once every 16 days, about once every 17 days,about once every 18 days, about once every 19 days, about once every 20days, about once every 21 days, about once every 22 days, about onceevery 23 days, about once every 24 days, about once every 25 days, aboutonce every 26 days, about once every 27 days, about once every 28 days,about once every 29 days, about once every 30 days, or about once every31 days.

The pharmaceutical compositions of the present invention mayalternatively be administered about once every week, about once every 2weeks, about once every 3 weeks, about once every 4 weeks, about onceevery 5 weeks, about once every 6 weeks, about once every 7 weeks, aboutonce every 8 weeks, about once every 9 weeks, about once every 10 weeks,about once every 11 weeks, about once every 12 weeks, about once every13 weeks, about once every 14 weeks, about once every 15 weeks, aboutonce every 16 weeks, about once every 17 weeks, about once every 18weeks, about once every 19 weeks, about once every 20 weeks.

Alternatively, the pharmaceutical compositions of the present inventionmay be administered about once every month, about once every 2 months,about once every 3 months, about once every 4 months, about once every 5months, about once every 6 months, about once every 7 months, about onceevery 8 months, about once every 9 months, about once every 10 months,about once every 11 months, or about once every 12 months.

Alternatively, the pharmaceutical compositions may be administered atleast once a week for about 2 weeks, at least once a week for about 3weeks, at least once a week for about 4 weeks, at least once a week forabout 5 weeks, at least once a week for about 6 weeks, at least once aweek for about 7 weeks, at least once a week for about 8 weeks, at leastonce a week for about 9 weeks, at least once a week for about 10 weeks,at least once a week for about 11 weeks, at least once a week for about12 weeks, at least once a week for about 13 weeks, at least once a weekfor about 14 weeks, at least once a week for about 15 weeks, at leastonce a week for about 16 weeks, at least once a week for about 17 weeks,at least once a week for about 18 weeks, at least once a week for about19 weeks, or at least once a week for about 20 weeks.

Alternatively the pharmaceutical compositions may be administered atleast once a week for about 1 month, at least once a week for about 2months, at least once a week for about 3 months, at least once a weekfor about 4 months, at least once a week for about 5 months, at leastonce a week for about 6 months, at least once a week for about 7 months,at least once a week for about 8 months, at least once a week for about9 months, at least once a week for about 10 months, at least once a weekfor about 11 months, or at least once a week for about 12 months.

Combination Therapy

In addition, co-administration or sequential administration of thecompounds of the present invention and other therapeutic agents may bedesirable, such as chemotherapeutic agents, immunosuppressive agents,cytokines, cytotoxic agents, nucleolytic compounds, radioactiveisotopes, receptors, and pro-drug activating enzymes, which may benaturally occurring or produced by recombinant methods. The combinedadministration includes co-administration, using separate formulationsor a single pharmaceutical formulation, and consecutive administrationin either order, wherein preferably there is a time period while both(or all) active therapeutic agents simultaneously exert their biologicalactivities.

The compounds of this invention may be administered in combination withat least one selected from the group consisting of an antirheumatic(e.g., methotrexate, auranofin, aurothioglucose, azathioprine,etanercept, gold sodium thiomalate, hydroxychloroquine sulfate,leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an anti-cancer, anantimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, anantiviral, a carbapenem, cephalosporin, a flurorquinolone, a macrolide,a penicillin, a sulfonamide, a tetracycline, another antimicrobial), ananti-psoriatic, a corticosteriod, an anabolic steroid, adiabetes-related agent, a mineral, a nutritional, a thyroid agent, avitamin, a calcium-related hormone, an antidiarrheal, an anti-tussive,an anti-emetic, an anti-ulcer, a laxative, an anticoagulant, anerythropieitin (e.g., epoetin alpha), a filgrastim (e.g., G-CSF,Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, animmunoglobulin, an immunosuppressive (e.g., basiliximab, cyclosporine,daclizumab), a growth hormone, a hormone replacement drug, an estrogenreceptor modulator, a mydriatic, a cycloplegic, an alkylating agent, ananti-metabolite, a mitotic inhibitor, a radiopharmaceutical, ananti-depressant, anti-manic agent, an anti-psychotic, an anxiolytic, ahypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthmamedication, a beta agonist, an inhaled steroid, a leukotriene inhibitor,a methylxanthine, a cromolyn, an epinephrine or analog thereof, dornasealpha (Pulmozyme), or a cytokine.

Such anti-cancer or antimicrobial compounds can also include toxinmolecules that are associated, bound, co-formulated, co-administered orsequentially administered, in either order, with at least one of thecompounds of the present invention. The toxin can optionally act toselectively kill the pathologic cell or tissue. The pathologic cell canbe a cancer or other cell. Such toxins can be, but are not limited to,purified or recombinant toxin or toxin fragment comprising at least onefunctional cytotoxic domain of toxin, e.g., selected from at least oneof ricin, diphtheria toxin, a venom toxin, or a bacterial toxin. Theterm toxin also includes both endotoxins and exotoxins produced by anynaturally occurring, mutant or recombinant bacteria or viruses which maycause any pathological condition in humans and other mammals, includingtoxin shock, which can result in death. Such toxins may include, but arenot limited to, enterotoxigenic E. coli heat-labile enterotoxin (LT),heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonasenterotoxins, toxic shock syndrome toxin-1 (TSST-1), Staphylococcalenterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal enterotoxins andthe like. Such bacteria include, but are not limited to, strains of aspecies of enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli(e.g., strains of serotype 0157:H7), Staphylococcus species (e.g.,Staphylococcus aureus, Staphylococcus pyogenes), Shigella species (e.g.,Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigellasonnei), Salmonella species (e.g., Salmonella typhi, Salmonellacholera-suis, Salmonella enteritidis), Clostridium species (e.g.,Clostridium perfringens, Clostridium dificile, Clostridium botulinum),Camphlobacter species (e.g., Camphlobacter jejuni, Camphlobacter fetus),Heliobacter species, (e.g., Heliobacter pylori), Aeromonas species(e.g., Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae),Pleisomonas shigelloides, Yersina enterocolitica, Vibrios species (e.g.,Vibrios cholerae, Vibrios parahemolyticus), Klebsiella species,Pseudomonas aeruginosa, and Streptococci. See, e.g., Stein, ed.,INTERNAL MEDICINE, 3rd ed., pp 1-13, Little, Brown and Co., Boston,(1990); Evans et al., eds., Bacterial Infections of Humans: Epidemiologyand Control, 2d. Ed., pp 239-254, Plenum Medical Book Co., New York(1991); Mandell et al, Principles and Practice of Infectious Diseases,3d. Ed., Churchill Livingstone, New York (1990); Berkow et al, eds., TheMerck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992; Wood etal, FEMS Microbiology Immunology, 76:121-134 (1991); Marrack et al,Science, 248:705-711 (1990), the contents of which references areincorporated entirely herein by reference.

More specifically, the compound of the present invention may beadministered in combination with at least one immunosuppressive agentfor use in, for example, treating or preventing a vascular occlusiveconditions such as transplant vasculopathy. Suitable immunosuppressiveagents include, but are not limited to, CellCept (Roche Labs.), Gengraf(Abbott Labs., Inc.), Micrhogam (Ortho-Clinical), Neoral (Novartis),Orthoclone OKT3 (Ortho-Biotech), Prograf (Fujisawa), Rapamune(Wyeth-Ayerst), Sandimmune (Novartis), Thymoglobulin (SangStat), Zenapax(Roche).

In one embodiment, the therapeutic agent administered simultaneously orsequentially, in either order and at various times with a compound ofthe present invention, comprises a chemotherapeutic agent. A“chemotherapeutic agent” is a compound useful in the treatment ofcancer. Examples of chemotherapeutic agents include, but are not limitedto, alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethylenethiophosphaoramide andtrimethylolomelamine; nitrogen mustards such as chlorambucil,chlomaphazine, cholophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembiehin, phenesterine, prednimustine, trofosfamide, uracil mustard;nitroureas such as cannustine, chlorozotocin, fotemustine, lomustine,nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, calicheamicin,carabicin, carminomycin, carzinophilin, chromoinycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,epirubicin, esorubicin, idambicin, marcellomycin, mitomycins,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues such asdenopterin, methotrexate, pteropterin, trimetrexate; purine analogs suchas fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofrran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.,paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included in this definition areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4 hydroxytamoxifen,trioxifene, keoxifene, onapristone, and toremifene (Fareston); andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

In another embodiment, the therapeutic agent comprises a cytokine. Theterm “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β; plateletgrowth factor; transforming growth factors (TGFs) such as TGF-α andTGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (GCSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis factor such asTNF-α or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

In another embodiment, the compounds of the present invention may beadministered in combination with an anti-inflammatory agent including,but not limited to, adrenocortical steroids (cortisol, cortisone,fludrocortisone, prednisone, prednisolone, 6α-methylprednisolone,triamcinolone, betamethasone, and dexamethasone), non-steroidal agents(salicylic acid derivatives, i.e., aspirin; para-aminophenolderivatives, i.e., acetominophen; indole and indene acetic acids(indomethacin, sulindac, and etodalac), heteroaryl acetic acids(tolmetin, diclofenac, and ketorolac), arylpropionic acids (ibuprofenand derivatives), anthranilic acids (mefenamic acid, and meclofenamicacid), enolic acids (piroxicam, tenoxicam, phenylbutazone, andoxyphenthatrazone), nabumetone, gold compounds (auranofin,aurothioglucose, gold sodium thiomalate). Commercially availablenonsteroidal anti-inflammatory drugs include, but are not limited to,Anaprox (Roche Labs.), Arthrotec (Searle), Cataflam (Novartis), Celebrex(Pfizer), Clinoril (Merck), Dolobid (Merck), Feldene (Pfizer), Indocin(Merck), Lodine (Wyeth-Ayerst), Mobic (Boehringer Ingelheim), Motrin(McNeil Consumer), Naprosyn (Roche Labs.), Orudis (Wyeth-Ayerst),Oruvail (Wyeth-Ayerst), Ponstel (First Horizon), Relafen(GlaxoSmithKline), Tolectin (Ortho-McNeil), Toradol (Roche Labs., Inc.),Vioxx (Merck), Voltaren (Novartis), Advair (GlaxoSmithKline), Flovent(GlaxoSmithKline), Pulmicort (AstranZeneca), and Vanceril (Schering),Asacol (Procter & Gamble), Colazal (Salix), Dipentum (Pharmacia &Upjohn), and Rowasa (Solvay).

In yet another embodiment, the compounds of the present invention may beadmistered in combination with an antirheumatic agent. Commerciallyavailable antirheumatic agents include, but are not limited to, Anaprox(Roche Labs.), Arava (Aventic), Arthrotec (Searle), Azulfidine(Pharmacia & Upjohn), Cataflam (Novartis), Celebrex (Pfizer), Celestone(Schering), Cuprimine (Merck), Enbrel (Immunex), Feldene (Pfizer),Gengraf (Abbott), Indocin (Merck), Lodine (Wyeth-Ayerst), Naprosyn(Roche Labs.), Neoral (Novartis), Pediapred (Celltech), Prednisone(Roxanne), Remicade (Centocor), Solu-Medrol (Pharmacia & Upjohn),Triliate (Purdue Frederick), and Voltaren (Novartis).

Moreover, the compounds of the present invention may be used incombination with any cardiovascular agent including, but not limited to,adrenergic blockers such as Cardura (Pfizer), Dibenzyline (WellSpring),Hytrin (Abbott), Minipress (Pfizer), and Minizide (Pfizer); adrenergicstimulants such as Aldoclor (Merck), Aldomet (Merck), Aldoril (Merck),Catapres (Boehringer Ingelheim), Clorpres (Bertek), and Tenex (Robins);alpha/beta adrenergic blockers such as Coreg (GlaxoSmithKline), andNormodyne (Schering); angiotensin converting enzyme inhibitors such asAccupril (Parke-Davis), Aceon (Solvay), Altace (Monarch), Captopril(Mylan), Enalaprilat (Baxter Anesthesia), Lotensin (Novartis), Mavik(Abbott), Monopril (Bristol-Myers Squibb), Prinivil (Merck), Univasc(Schwarz), Vaotec (Merck), and Zestril (AstraZeneca); angiotenisinconverting enzyme inhibitors such as Lexxel (AstraZeneca), Lotrel(Novartis), Tarka (Abbott), Accuretic (Parke-Davis), Lotensin(Novartis), Prinzide (Merck), Uniretic (Schwarz), Vaeretic (Merck), andZestoretic (AstraZeneca); angiotensin II receptor antagonists such asAtacand (AstraZeneca), Avapro (Briston-Myers Squibb), Cozaar (Merck),Diovan (Novartis), Micardis (Boehringer Ingelheim), and Teveten(Unimed); antiarrhythmics (Groups I-IV), antilipemic agents such as bileacid sequestrants, fibric acid derivatives, HMG-CoA reductaseinhibitors, and nicotinic acid; Beta adrenergic blocking agents; calciumchannel blockers; inotropic agents; vasodilators including coronoaryvasodilators, natriuretic peptides, and peripheral vasodilators; andvasopressors.

In another aspect of the present invention, the therapeutic agentcomprises a small molecule toxin, including maytansine, calicheamicin,trichothene, and CC 1065. In a specific embodiment, the therapeuticagent may comprise one more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. Structured analogues ofcalicheamicin are also known. See Hinman et al., 53 CANCER RESEARCH3336-42 (1993); Lode et al., 58 CANCER RESEARCH 2925-28 (1998).

In yet another aspect of the present invention, the therapeutic agentmay comprise one or more enzymatically active toxins and fragmentsthereof. Examples of such toxins include nonbinding active fragments ofdiphtheria toxin, diphtheria A chain, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, dianthin proteins, Phytolaca americana proteins (PAPI,PAPAII, and PAP-S), momordica charantia inhibitor, curcin, crotinsapaonaria officinalis inhibitor, gelonin, mitogellin, restrictoein,phenomycin, enomycin and the tricothecenes. See, e.g., WO 93/21232.

The present invention further contemplates therapeutic agents that havenucleolytic activity such as a ribonuclease and a deoxyribonuclease. Inaddition, a variety of radioactive isotopes are available for theproduction of radioconjugated binding partners. Examples include Y⁹⁰,At²²², Ret⁸⁶, Re¹⁸⁶, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu.

In yet another aspect of the present invention, the at least onecompound may be conjugated to a receptor, such as streptavidin, forutilization in tumor pretargeting. Briefly, the compound-receptorconjugate is administered to the patient and unbound conjugate isremoved from circulation with a clearing agent. A ligand, such asbiotin, which is conjugated to a cytotoxic agent is then administered.

Timing of Administration

In several embodiments of the present invention, a compound describedherein is administered before or after administration of a secondtherapeutic agent. The administration of a compound may occur anytimefrom several minutes to several hours before the administration of thesecond therapeutic agent. The compound may alternatively be administeredanytime from several hours to several days, possibly several weeks, andup to several months before the second therapeutic agent.

More specifically, a compound of the present invention may beadministered at least about 1 minute, at least about minutes, at leastabout minutes, at least about minutes, at least about minutes, at leastabout 2 minutes, at least about 3 minutes, at least about 4 minutes, atleast about 5 minutes, at least about 6 minutes, at least about 7minutes, at least about 8 minutes, at least about 9 minutes, at leastabout 10 minutes, at least about 11 minutes, at least about 12 minutes,at least about 13 minutes, at least about 14 minutes, at least about 15minutes, at least about 16 minutes, at least about 17 minutes, at leastabout 18 minutes, at least about 19 minutes, at least about 20 minutes,at least about 21 minutes, at least about 22 minutes, at least about 23minutes, at least about 24 minutes, at least about 25 minutes, at leastabout 26 minutes, at least about 27 minutes, at least about 28 minutes,at least about 29 minutes, at least about 30 minutes, at least about 31minutes, at least about 32 minutes, at least about 33 minutes, at leastabout 34 minutes, at least about 35 minutes, at least about 36 minutes,at least about 37 minutes, at least about 38 minutes, at least about 39minutes, at least about 40 minutes, at least about 41 minutes, at leastabout 42 minutes, at least about 43 minutes, at least about 44 minutes,at least about 45 minutes, at least about 46 minutes, at least about 47minutes, at least about 48 minutes, at least about 49 minutes, at leastabout 50 minutes, at least about 51 minutes, at least about 52 minutes,at least about 53 minutes, at least about 54 minutes, at least about 55minutes, at least about 56 minutes, at least about 57 minutes, at leastabout 58 minutes, at least about 59 minutes, or at least about 60minutes before or after the second therapeutic agent. Furthermore, acompound of the present invention may be administered at least about 1hour, at least about 2 hours, at least about 3 hours, at least about 4hours, at least about 5 hours, at least about 6 hours, at least about 7hours, at least about 8 hours, at least about 9 hours, at least about 10hours, at least about 11 hours, at least about 12 hours, at least about13 hours, at least about 14 hours, at least about 15 hours, at leastabout 16 hours, at least about 17 hours, at least about 18 hours, atleast about 19 hours, at least about 20 hours, at least about 21 hours,at least about 22 hours, at least about 23 hours, or at least about 24hours before or after the second therapeutic agent.

Moreover, a compound of the present invention may be administered atleast about 1 day, at least about 2 days, at least about 3 days, atleast about 4 days, at least about 5 days, at least about 6 days, atleast about 7 days, at least about 8 days, at least about 9 days, atleast about 10 days, at least about 11 days, at least about 12 days, atleast about 13 days, at least about 14 days, at least about 15 days, atleast about 16 days, at least about 17 days, at least about 18 days, atleast about 19 days, at least about 20 days, at least about 21 days, atleast about 22 days, at least about 23 days, at least about 24 days, atleast about 25 days, at least about 26 days, at least about 27 days, atleast about 28 days, at least about 29 days, at least about 30 days orat least about 31 days before or after the administration of the secondtherapeutic agent.

In yet another aspect of the present invention, a compound of thepresent invention may be administered at least about 1 week, at leastabout 2 weeks, at least about 3 weeks, at least about 4 weeks, at leastabout 5 weeks, at least about 6 weeks, at least about 7 weeks, at leastabout 8 weeks, at least about 9 weeks, at least about 10 weeks, at leastabout 11 weeks, at least about 12 weeks, at least about 13 weeks, atleast about 14 weeks, at least about 15 weeks, at least about 16 weeks,at least about 17 weeks, at least about 18 weeks, at least about 19weeks, or at least about 20 weeks before or after the second therapeuticagent.

In a further aspect of the present invention, a compound of the presentinvention may be administered at least about one month, at least abouttwo months, at least about three months, at least about four months, atleast about five months, at least about six months, at least about sevenmonths, at least about eight months, at least about nine months, atleast about ten months, at least about eleven months, or at least abouttwelve months before or after the second therapeutic agent.

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.

Definitions

As used herein, the term “compound” includes both the singular and theplural, and includes any single entity or combined entities that have atleast the activity disclosed herein and combinations, fragments, analogsor derivatives of such entities. Such entities include, but are notlimited to, chemical elements, molecules, compounds, mixtures,emulsions, chemotherapeutic agents, pharmacological agents, hormones,antibodies, growth factors, cellular factors, nucleic acids, proteins,peptides, peptidomimetics, nucleotides, carbohydrates, and combinations,fragments, analogs or derivatives of such entities.

The term “phenylamine” refers to a primary or secondary benzeneamine,more commonly known as an aniline. The amino group on the aniline can besubstituted with hydrogen, alkyl (C₁-C₁₂, straight chain or branched),cycloalkyl (C₃-C₁₀), or aryl substituted aryl groups. The phenyl ring ofthis aniline derivative can be optionally substituted with one or morefunctional groups, or a combination of functional groups such as alkyl,alkenyl, alkynyl, phenyl, benzyl, halo, cyano, nitro, hydroxy, thioxy,alkoxy, aryloxy, haloalkyloxy, alkylthio, arylthio, amino, alkyl amino,aryl amino, acyl, carboxyl, amido, sulfonamido, sulfonyl, sulfate,sulfonic acid, morpholino, piperazinyl, pyridyl, thienyl, furanyl,pyrroyl, pyrazoyl, phosphate, phosphonic acid, or phosphonate. Ifapplicable, these groups can be represented in protected or unprotectedforms used in standard organic synthesis.

The term “naphthylamine” refers to a primary or secondary α- orβ-naphthylamine. The ring substructure in the naphthylamine can beoptionally substituted with one or a combination of functional groupssuch as alkyl, alkenyl, alkynyl, phenyl, benzyl, halo, cyano, nitro,hydroxy, thioxy, alkoxy, aryloxy, haloalkyloxy, alkylthio, arylthio,amino, alkyl amino, aryl amino, acyl, carboxyl, amido, sulfonamido,sulfonyl, sulfate, sulfonic acid, morpholino, thiomorpholino,piperazinyl, pyridyl, thienyl, furanyl, pyrroyl, pyrazoyl, phosphate,phosphonic acid, phosphonate and the like. These groups can berepresented in protected or unprotected forms used in standard organicsynthesis.

The term “naphthylalkyl amine” refers to a primary or secondary α- andβ-naphthylalkyl amine (for example, 2-α-naphthylethyl amine). The term“benzalkyl amine” refers to a primary or secondary benzylalkyl amine(for example, phenylethyl amine). These aryl alkyl substructures orcompounds can be optically active or optically inactive. The aryl (ring)substructures of the naphthylalkyl and benzalkyl amines can beoptionally subsituted with one or a combination of functional groups,such as alkyl, alkenyl, alkynyl, phenyl, benzyl, halo, cyano, nitro,hydroxy, thioxy, alkoxy, aryloxy, haloalkyloxy, alkylthio, arylthio,amino, alkyl amino, aryl amino, acyl, carbolyl, amido, sulfonamido,sulfonyl, sulfate, sulfonic acid, morpholino, piperazinyl, pyridyl,thienyl, furanyl, pyrroyl, pyrazoyl, phosphate, phosphonic acid,phosphonate and the like. If applicable these groups can be representedin protected or unprotected forms used in standard organic synthesis.

The term “quinolinyl amine” refers to primary or secondary quinolylamines. These amines can be in optically active or inactive forms. Thearyl (ring) substructure of the quinolyl amine can be be optionallysubstituted with one a combination of functional groups such as alkyl,alkenyl, alkynyl, phenyl, benzyl, halo, cyano, nitro, hydroxy, thioxy,alkoxy, aryloxy, haloalkyloxy, alkylthio, arylthio, amino, alkyl amino,aryl amino, acyl, carboxyl, amido, sulfonamido, sulfonyl, sulfate,sulfonic acid, morpholino, thiomorpholino, piperazinyl, pyridyl,thienyl, furanyl, pyrroyl, pyrazoyl, phosphate, phosphonic acid,phosphonate and the like. These groups can be represented in protectedor unprotected forms used in standard organic synthesis.

The term “heteroaryl amines” refers to pyrroles, pyrazoles, imidazoles,and indoles. The aryl (ring) substructure of the heteroaryl amine can beoptionally substituted with one or a combination of functional groupssuch as alkyl, alkenyl, alkynyl, phenyl, benzyl, halo, cyano, nitro,hydroxy, thioxy, alkoxy, aryloxy, haloalkyloxy, alkylthio, arylthio,amino, alkyl amino, aryl amino, acyl, carboxyl, amido, sulfonamido,sulfonyl, sulfate, sulfonic acid, morpholino, thiomorpholino,piperazinyl, phosphate, phosphonic acid, or phosphonate. These groupscan be represented in protected or unprotected forms used in standardorganic synthesis.

The term “glycated protein,” as used herein, includes proteins linked toglucose, either enzymatically or non-enzymatically, primarily bycondensation of free epsilon-amino groups in the protein with glucose,forming Amadori adducts. Furthermore, glycated protein, as used herein,includes not only proteins containing these initial glycation products,but also glycation products resulting from further reactions such asrearrangements, dehydration, and condensations that form irreversibleadvanced glycation end products (AGE).

The term “polynucleotide” refers generally to polymeric forms ofnucleotides of any length, either ribonucleotides or deoxynucleotides.Thus, this term includes, but is not limited to, single-stranded,double-stranded, or multi-stranded DNA or RNA. Polynucleotides mayfurther comprise genomic DNA, cDNA, or DNA-RNA hybrids. Moreover, thepolynucleotides of the present invention may be synthetically produced.

Polynucleotides may comprise chemically modified, biochemicallymodified, or derivatized nucleotides. For example, a polynucleotide maycomprise, in part, modified nucleotides such as methylated nucleotidesor nucleotide analogs. In other embodiments, polynucleotides maycomprise sugars, caps, nucleotide branches, and linking groups such asfluororibose and thioate. In addition, the sequence of nucleotides maybe interrupted by non-nucleotide components. Furthermore, apolynucleotide may be modified after polymerization to facilitate itsattachment to other polynucleotides, proteins, metal ions, labelingcomponents, or a solid support.

The backbone of the polynucleotide may comprise modified or substitutedsugar and/or phosphate groups. Alternatively, the backbone of thepolynucleotide may comprise a polymer of synthetic subunits such asphosphoramidites and thus may be an oligodeoxynucleoside phosphoramidateor a mixed phosphoramidate-phosphodiester oligomer. See Peyrottes etal., NUCL. ACIDS RES. (1996) 24:1841-1848, and Chaturvedi et al., NUCL.ACIDS RES. (1996) 24:2318-2323.

The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget polynucleotide; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

The term “gene” refers to a polynucleotide sequence that comprisescoding sequences necessary for the production of a polypeptide orprecursor, and may also include expression control sequences or othercontrol or regulatory sequences. The polypeptide can be encoded by afull length coding sequence or by any portion of the coding sequence.The gene may be derived in whole or in part from any source known tothose of ordinary skill in the art including a plant, a fungus, ananimal, a bacterial genome or episome, eukaryotic, nuclear or plasmidDNA, cDNA, viral DNA, or chemically synthesized DNA. A gene mayconstitute an uninterrupted coding sequence or it may include one ormore introns, bound by the appropriate splice junctions. Moreover, agene may contain one or more modifications in either the coding or theuntranslated regions that could affect certain properties of thepolynucleotide or polypeptide, such as the biological activity or thechemical structure of the expression product, the rate of expression, orthe manner of expression control. Such modifications include, but arenot limited to, mutations, insertions, deletions, and substitutions ofone or more nucleotides. In this regard, such modified genes may bereferred to as variants of the native gene.

“Gene expression” refers to the process by which a polynucleotidesequence undergoes successful transcription and translation such thatdetectable levels of the nucleotide sequence are expressed as proteinsor the polynucleotide sequence undergoes transcription, if RNA is copiedfrom DNA, or replication if DNA is copied from DNA, such that theresulting nucleotide copies are detectable.

The term “gene expression profile” refers to a group of genesrepresenting a particular cell or tissue type (e.g., neuron, coronaryartery endothelium, or disease tissue) in any activation state. In oneaspect, a gene expression profile is generated from cells exposed to acompound of the present invention. This profile may be compared to agene expression profile generated from the same type of cell or tissuetype prior to treatment with a compound of the present invention.Furthermore, a series of gene expression profiles may be generated fromcells or tissues treated with a compound of the present invention,specifically, at different doses or a time-course to assess the effectsof the compound. A gene expression profile is also known as a geneexpression signature.

The term “differential expression” refers to both quantitative as wellas qualitative differences in the temporal and tissue expressionpatterns of a gene. For example, a differentially expressed gene mayhave its expression activated or completely inactivated in normal versusdisease conditions. Such a qualitatively regulated gene may exhibit anexpression pattern within a given tissue or cell type that is detectablein either control or disease conditions, but is not detectable in both.“Differentially expressed polynucleotide,” as used herein, refers to apolynucleotide sequence that uniquely identifies a differentiallyexpressed gene so that detection of the differentially expressedpolynucleotide in a sample is correlated with the presence of adifferentially expressed gene in a sample.

Similarly, a differentially expressed protein may have its expressionactivated or completely inactivated in normal versus disease conditions.Such a qualitatively regulated protein may exhibit an expression patternwithin a given tissue or cell type that is detectable in either controlor disease conditions, but is not detectable in both. A “differentiallyexpressed protein,” as used herein, refers to an amino acid sequencethat uniquely identifies a differentially expressed protein so thatdetection of the differentially expressed protein in a sample iscorrelated with the presence of a differentially expressed protein in asample.

“Cell type,” as used herein, refers to a cell from a given source (e.g.,tissue or organ), a cell in a given state of differentiation, or a cellassociated with a given pathology or genetic makeup.

The term “polypeptide” refers to a polymeric form of amino acids of anylength, which may include translated, untranslated, chemically modified,biochemically modified, and derivatized amino acids. A polypeptide maybe naturally occurring, recombinant, or synthetic, or any combination ofthese. Moreover, the term “polypeptide,” as used herein, refers toproteins, polypeptides, and peptides of any size, structure, orfunction. For example, a polypeptide may comprise a string of aminoacids held together by peptide bonds. A polypeptide may alternativelycomprise a long chain of amino acids held together by peptide bonds.Moreover, a polypeptide may also comprise a fragment of a naturallyoccurring protein or peptide. A polypeptide may be a single molecule ormay be a multi-molecular complex. In addition, such polypeptides mayhave modified peptide backbones as well.

The term “polypeptide” further comprises immunologically tagged proteinsand fusion proteins, including, but not limited to, fusion proteins witha heterologous amino acid sequence, fusion proteins with heterologousand homologous leader sequences, and fusion proteins with or withoutN-terminal methionine residues.

The term “protein expression” refers to the process by which apolynucleotide sequence undergoes successful transcription andtranslation such that detectable levels of the amino acid sequence orprotein are expressed.

The term “protein expression profile” refers to a group of proteinsrepresenting a particular cell or tissue type (e.g., neuron, coronaryartery endothelium, or disease tissue). In one aspect, a proteinexpression profile is generated from cells or tissues exposed to acompound of the present invention. This profile may be compared to aprotein expression profile generated from the same type of cell ortissue prior to treatment with a compound of the present invention.Furthermore, a series of protein expression profiles may be generatedfrom cells or tissues treated with a compound of the present invention,specifically, at different doses or a time-course to assess the effectsof the compound. A protein expression profile is also known as a“protein expression signature.”

As used herein, a “biomolecule” includes polynucleotides andpolypeptides. Moreover, a “biomolecular sequence,” as used herein, is aterm that refers to all or a portion of a polynucleotide sequence. Abiomolecular sequence may also refer to all or a portion of apolypeptide sequence. In the context of biomolecule, for example,perlecan, the term “functional equivalent” refers to a protein orpolynucleotide molecule that possesses functional or structuralcharacteristics that are substantially similar to all or part of thenative perlecan protein or native perlecan-encoding polynucleotides. Afunctional equivalent of a native perlecan protein may containmodifications depending on the necessity of such modifications for aspecific structure or the performance of a specific function. The term“functional equivalent” is intended to include the “fragments,”“mutants,” “derivatives,” “alleles,” “hybrids,” “variants,” “analogs,”or “chemical derivatives” of native perlecan.

A “host cell,” as used herein, refers to a microorganism, a prokaryoticcell, a eukaryotic cell or cell line cultured as a unicellular entitythat may be, or has been, used as a recipient for a recombinant vectoror other transfer of polynucleotides, and includes the progeny of theoriginal cell that has been transfected. It is understood that theprogeny of a single cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement as the original parentdue to natural, accidental, or deliberate mutation.

In the context of immunoglobulins, the term “functional equivalent”refers to immunoglobulin molecules that exhibit immunological bindingproperties that are substantially similar to the parent immunoglobulin.As used herein, the term “immunological binding properties” refers tonon-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. Indeed, a functional equivalent of a monoclonal antibodyimmunoglobulin, for example, may inhibit the binding of the parentmonoclonal antibody to its antigen. A functional equivalent may compriseF(ab′)₂ fragments, F(ab) molecules, Fv fragments, single chain fragmentvariable displayed on phage (scFv), single domain antibodies, chimericantibodies, or the like so long as the immunoglobulin exhibits thecharacteristics of the parent immunoglobulin.

As used herein, the term “isolated” refers to a polynucleotide, apolypeptide, an antibody, or a host cell that is in an environmentdifferent from that in which the polynucleotide, the polypeptide, theantibody, or the host cell naturally occurs. An isolated polynucleotide,polypeptide, antibody, or host cell is generally substantially purified.

As used herein, the term “substantially purified” refers to a compoundthat is removed from its natural environment and is at least about 60%to 99.9% free from other components, or is at least about 60% free, atleast about 65% free, at least about 70% free, at least about 75% free,at least about 80% free, at least about 83% free, at least about 85%free, at least about 88% free, at least about 90% free, at least about91% free, at least about 92% free, at least about 93% free, at leastabout 94% free, at least about 95% free, at least about 96% free, atleast about 97% free, at least about 98% free, at least about 99% free,at least about 99.9% free, or at least about 99.99% free from othercomponents with which it is naturally associated. For example, acomposition containing A is “substantially free of” B when at leastabout 85% by weight of the total A+B in the composition is A.Alternatively, A comprises at least about 90% by weight of the total ofA+B in the composition, further still, at least about 95% or even 99% byweight.

“Diagnosis,” as used herein, generally includes a determination of asubject's susceptibility to a disease or disorder, a determination as towhether a subject is presently affected by a disease or disorder, aprognosis of a subject affected by a disease or disorder (e.g.,identification of pre-metastatic or metastatic cancerous states, stagesof cancer, or responsiveness of cancer to therapy), and therametrics(e.g., monitoring a subject's condition to provide information as to theeffect or efficacy of therapy).

The term “biological sample” encompasses a variety of sample typesobtained from or originating from an organism which may be used indiagnostic, monitoring, or other assays. The term encompasses blood,serum, plasma, cells, proteins, carbohydrates, nucleic acids, urine,nasal secretions, mucosal secretions, cellular fluid, cellular exudateand other liquid samples of biological origin, solid tissue samples suchas a biopsy specimen, or tissue cultures or cells derived therefrom andthe progeny thereof. The term specifically encompasses a clinicalsample, and further includes cells in cell culture, cell supernatants,cell lysates, amniotic fluid, biological fluids, and tissue samples. Theterm also encompasses samples that have been manipulated in any wayafter procurement such as treatment with reagents, solubilization, orenrichment for certain components. The biological sample can be derivedfrom the organism directly or can be collected from the environment.

The terms “individual,” “subject,” “host,” and “patient” refer to anysubject for whom diagnosis, treatment, or therapy is desired. In oneembodiment, the individual, subject, host, or patient is a human. Othersubjects may include, but are not limited to, animals including but notlimited to, cattle, sheep, horses, dogs, cats, guinea pigs, rabbits,rats, primates, opossums and mice. Other subjects include species ofbacteria, phages, cell cultures, viruses, plants and other eucaryotes,prokaryotes and unclassified organisms.

The terms “treatment,” “treating,” “treat,” and the like are used hereinto refer generally to obtaining a desired pharmacological and/orphysiological effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in asubject, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom, but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

The expression “therapeutically effective amount” refers to an amountof, for example, a compound disclosed herein, that is effective forpreventing, ameliorating, treating or delaying the onset of a disease orcondition.

A “prophylactically effective amount” refers to an amount of, forexample, a compound disclosed herein that is effective for preventing adisease or condition.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant, which is useful for delivery of a drugto a subject, such as a mammal or other animal. The compounds of thepresent invention may be delivered by a liposome. The components of theliposome are commonly arranged in a bilayer formation, similar to thelipid arrangement of biological membranes. Liposome formulations,loading of liposomes and administration and delivery of liposomes areknown in the art.

“Hybridization,” broadly defined, refers to any process by which apolynucleotide sequence binds to a complementary sequence through basepairing. Hybridization conditions can be defined by, for example, theconcentrations of salt or formamide in the prehybridization andhybridization solutions, or by the hybridization temperature, and arewell known in the art. Hybridization can occur under conditions ofvarious stringency. Hybridization may also refer to the binding of aprotein-capture agent to a target protein under certain conditions, suchas normal physiological conditions.

As understood herein, the term “activation” refers to any alteration ofa signaling pathway or biological response including, for example,increases above basal levels, restoration to basal levels from aninhibited state, and stimulation of the pathway above basal levels.

The term “biological activity” refers to the biological behavior andeffects of a protein or peptide. The biological activity of a proteinmay be affected at the cellular level and the molecular level. Forexample, an antisense oligonucleotide may prevent translation of aparticular mRNA, thereby inhibiting the biological activity of theprotein encoded by the mRNA. In addition, an antibody may bind to aparticular protein and inhibit that protein's biological activity.

The term “oligonucleotide” as used herein refers to a polynucleotidesequence comprising, for example, from about 4 nucleotides (nt) to about1000 nt. Oligonucleotides for use in the present invention arepreferably from about 15 nt to about 150 nt, more preferably from about150 nt to about 1000 nt in length. The oligonucleotide may be anaturally occurring oligonucleotide or a synthetic oligonucleotide.Oligonucleotides may be prepared by the phosphoramidite method (Beaucageand Carruthers, TETRAHEDRON LETT. (1981) 22:1859-1862), or by thetriester method (Matteucci et al., J. AM. CHEM. Soc. (1981) 103:3185),or by other chemical methods known in the art.

The term “microarray” refers generally to the type of genes or proteinsrepresented on a microarray by oligonucleotides (polynucleotidesequences) or protein-binding agents, and where the type of genes orproteins represented on the microarray is dependent on the intendedpurpose of the microarray (e.g., to monitor expression of human genes orproteins). The oligonucleotides or protein-binding agents on a givenmicroarray may correspond to the same type, category, or group of genesor proteins. Genes or proteins may be considered to be of the same typeif they share some common characteristics such as species of origin(e.g., human, mouse, rat); disease state (e.g., cancer); function (e.g.,protein kinases, tumor suppressors); same biological process (e.g.,apoptosis, signal transduction, cell cycle regulation, proliferation,differentiation). For example, one microarray type may be a “cancermicroarray” in which each of the microarray oligonucleotides orprotein-binding agents correspond to a gene or protein associated with acancer. An “epithelial microarray” may be a microarray ofoligonucleotides or protein-binding agents corresponding to uniqueepithelial genes or proteins. Similarly, a “cell cycle microarray” maybe an microarray type in which the oligonucleotides or protein-bindingagents correspond to unique genes or proteins associated with the cellcycle.

The term “detectable”, one in sense, refers to a polynucleotideexpression pattern which is detectable via the standard techniques ofpolymerase chain reaction (PCR), reverse transcriptase (RT)-PCR(RT-PCR), differential display, and Northern analyses, which are wellknown to those of skill in the art. Similarly, polypeptide expressionpatterns may be “detected” via standard techniques includingimmunoassays such as Western blots. In general, the term “detectable isused when a result of an action, such as addition of a compound in anassay step, is observable, particularly by physical means, such as acolor change.

A “target gene” refers to a polynucleotide, often derived from abiological sample, to which an oligonucleotide probe is designed tospecifically hybridize. It is either the presence or absence of thetarget polynucleotide that is to be detected, or the amount of thetarget polynucleotide that is to be quantified. The targetpolynucleotide has a sequence that is complementary to thepolynucleotide sequence of the corresponding probe directed to thetarget. The target polynucleotide may also refer to the specificsubsequence of a larger polynucleotide to which the probe is directed orto the overall sequence (e.g., gene or mRNA) whose expression level itis desired to detect.

A “target protein” refers to an polypeptide, often derived from abiological sample, to which a protein-capture agent specificallyhybridizes or binds. It is either the presence or absence of the targetprotein that is to be detected, or the amount of the target protein thatis to be quantified. The target protein has a structure that isrecognized by the corresponding protein-capture agent directed to thetarget. The target protein or amino acid may also refer to the specificsubstructure of a larger protein to which the protein-capture agent isdirected or to the overall structure (e.g., gene or mRNA) whoseexpression level it is desired to detect.

The term “complementary” refers to the topological compatibility ormatching together of the interacting surfaces of a probe molecule andits target. The target and its probe can be described as complementary,and furthermore, the contact surface characteristics are complementaryto each other. Hybridization or base pairing between nucleotides ornucleic acids, such as, for example, between the two strands of adouble-stranded DNA molecule or between an oligonucleotide probe and atarget are complementary.

The term “background” refers to non-specific binding or otherinteractions between, for example, polynucleotides, polypeptides, smallmolecules and polypeptides, or small molecules and polynucleotides.“Background” may also refer to the non-specific binding or otherinteractions in the context of assays including immuno assays.

In the context of microarrays, the term “background” refers tohybridization signals resulting from non-specific binding, or otherinteractions, between the labeled target polynucleotides and componentsof the oligonucleotide microarray (e.g., the oligonucleotide probes,control probes, the microarray support) or between target proteins andthe protein-binding agents of a protein microarray. Background signalsmay also be produced by intrinsic fluorescence of the microarraycomponents themselves. A single background signal may be calculated forthe entire microarray, or a different background signal may becalculated for each target polynucleotide or target protein. Thebackground may be calculated as the average hybridization signalintensity, or where a different background signal is calculated for eachtarget gene or target protein. Alternatively, background may becalculated as the average hybridization signal intensity produced byhybridization to probes that are not complementary to any sequence foundin the sample (e.g., probes directed to polynucleotides of the oppositesense or to genes not found in the sample such as bacterial genes wherethe sample is mammalian polynucleotides). The background can also becalculated as the average signal intensity produced by regions of themicroarray which lack any probes or protein-binding agents at all.

A “small molecule” comprises a compound or molecular complex, eithersynthetic, naturally derived, or partially synthetic, composed ofcarbon, hydrogen; oxygen, and nitrogen, which may also contain otherelements, and which may have a molecular weight of less than about 100to about 15,000 Daltons, or less than about 15,000, less than about14,000, less than about 13,000, less than about 12,000, less than about11,000, less than about 10,000, less than about 9,000, less than about8,000, less than about 7,000, less than about 6,000, less than about5,000, less than about 4,000, less than about 3,000, less than about2,000, less than about 1,000, less than about 900, less than about 800,less than about 700, less than about 600, less than about 500, less thanabout 400, less than about 300, less than about 200, or less than about100.

The term “fusion protein” refers to a protein composed of two or morepolypeptides that, although typically not joined in their native state,are joined by their respective amino and carboxyl termini through apeptide linkage to form a single continuous polypeptide. It isunderstood that the two or more polypeptide components can either bedirectly joined or indirectly joined through a peptide linker/spacer.

The term “normal physiological conditions” means conditions that aretypical inside a living organism or a cell. Although some organs ororganisms provide extreme conditions, the intra-organismal andintra-cellular environment normally varies around pH 7 (i.e., from pH6.5 to pH 7.5), contains water as the predominant solvent, and exists ata temperature above 0° C. and below 50° C. The concentration of varioussalts depends on the organ, organism, cell, or cellular compartment usedas a reference.

The term “cluster” refers to a group of clones or biomolecular sequencesrelated to one another by sequence homology. In one example, clustersare formed based upon a specified degree of homology and/or overlap(e.g., stringency). “Clustering” may be performed with the sequencedata. For instance, a biomolecular sequence thought to be associatedwith a particular molecular or biological activity in one tissue mightbe compared against another library or database of sequences. This typeof search is useful to look for homologous, and presumably functionallyrelated, sequences in other tissues or samples, and may be used tostreamline the methods of the present invention in that clustering maybe used within one or more of the databases to cluster biomolecularsequences prior to performing a method of the invention. The sequencesshowing sufficient homology with the representative sequence areconsidered part of a “cluster.” Such “sufficient” homology may varywithin the needs of one skilled in the art.

As used herein, the term “internal database” refers to a databasemaintained within a local computer network. It contains, for example,biomolecular sequences associated with a project. It may also containinformation associated with sequences including, but not limited to, alibrary in which a given sequence is found and descriptive informationabout a likely gene associated with the sequence. The internal databasemay typically be maintained as a private database behind a firewallwithin an enterprise network. However, the invention is not limited toonly this embodiment and an internal database could be made available tothe public. The internal database may include sequence data generated bythe same enterprise that maintains the database, and may also includesequence data obtained from external sources.

The term “external database,” as understood herein, refers to a databaselocated outside all internal databases. Typically, an enterprise networkdiffering from the enterprise network maintaining the internal databasewill maintain an external database. The external database may be used,for example, to provide some descriptive information on biomolecularsequences stored in the internal database. In one embodiment, theexternal database is GenBank and associated databases maintained by theNational Center for Biotechnology Information (NCBI), part of theNational Library of Medicine.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “compound” is a referenceto one or more such compounds and includes equivalents thereof known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed above and throughout the text areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

EXAMPLES

The present invention is further illustrated by the following examples,which are not to be construed in any way as imposing limitations uponthe scope thereof, but rather are illustrative only. On the contrary, itis to be clearly understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which, after readingthe description herein, may suggest themselves to one of ordinary skillin the art without departing from the spirit of the present invention orthe scope of the appended claims.

The following acronyms, abbreviations, terms and definitions have beenused throughout the experimental section. Acronyms or abbreviations:DIEA (N,N-diisopropylethylamine), THF (tetrahydrofuran), HPLC (highperformance liquid chromatography), TLC (thin layer chromatography), mp(melting point), rt (room temperature), aq (aqueous), min (minute), h(hr, hour), atm (atmosphere), conc. (concentrated), MS (massspectroscopy/spectrometry), NMR (nuclear magnetic resonance), R_(f) (TLCretention factor), and R_(t) (HPLC retention time). NMR abbreviations:br (broad), apt (apparent), s (singlet), d (doublet), t (triplet), q(quartet), dq (doublet of quartets), dd (doublet of doublets), dt(doublet of triplets), m (multiplet).

Example 1 General Synthetic, Purification, Characterization, andSpectroscopic Procedures

General Synthetic Procedures. Room temperature is defined as an ambienttemperature range, typically 20-25° C. An ice bath (crushed ice/water)temperature is defined as a range, typically −5 to 0° C. Temperature atreflux is defined as ±15° C. of the boiling point of the primaryreaction solvent. Overnight is defined as a time range of 8-16 hours.Vacuum filtration (water aspirator) is defined as range of 5-15 mm Hg.Dried under vacuum is defined as using a high vacuum pump as a range of0.1-5 mm Hg. Neutralization is defined as a typical acid-basedneutralization method and measured to a pH 6-8 range using pH-indicatingpaper. Brine is defined as a saturated aqueous sodium chloride. Nitrogenatmosphere is defined as positive static pressure of nitrogen gas passedthrough a Drierite column with an oil bubbler system. Concentratedammonium hydroxide is defined as an approximately 15 M solution.

All eluents for column or thin layer chromatography were prepared andreported as volume:volume (v:v) solutions, and HPLC eluent ratios arev:v ratios. Aqueous sodium hydroxide or sodium bicarbonate solutionswere prepared as weight:volume (w:v) ratios. Aqueous hydrochloric acidsolutions were prepared as v:v ratios.

The quantities of solvents and/or reagents used for reaction work-up orproduct isolation are those typically used by one trained in the art oforganic chemical synthesis, and the quantity of these solvents and/orreagents used is determined based upon synthetic experience andappropriateness to the specific reaction. For example: 1) crushed icequantity ranged from about 10-1000 g depending on reaction scale, 2)silica gel quantity used in column chromatography depended on materialquantity, complexity of mixture, and size of chromatography columnemployed and ranged from about 5-1000 g, 3) extraction solvent volumeranged from about 10-500 mL depending on reaction size, 4) washesemployed in compound isolation ranged from about 10-100 mL of solvent oraq reagent depending on scale of reaction 5) drying reagents (potassiumcarbonate, sodium carbonate or magnesium sulfate) ranged from about5-100 g depending on the amount of solvent to be dried and its watercontent.

Melting points were measured against a mercury thermometer and are notcorrected.

For column chromatography employing concentrated ammonium hydroxide aspart of the mobile phase, the fractions collected from the column weredried over sodium sulfate, potassium carbonate or a mixture of both.Then the organic layer was filtered by gravity or vacuum to remove thedrying agent prior to concentration/evaporation.

Flash Chromatography. In the Tables, “ISCO” indicates purification byflash chromatography as follows. Instrument: ISCO CombiFlash{umlaut over(S)}ï10×. Column: ISCO RediSepä—Disposable Columns for FlashChromatography (10 g of silica gel—normal phase—35-60 micron particlesize (230-400 mesh)). Mobile Phase A: CH₂Cl₂; Mobile Phase B: 10% NH₄OHin MeOH; Gradient: 0-10% B in 22 min, hold 10% B for 18 min; Fractions:30 fractions collected per column, 1.5 min each. Flow rate: 8.93 mL/min.The salient fractions were analyzed by MS and TLC (90:9:1CH₂Cl₂:MeOH:NH₄OH—R_(f) range 0.15-0.45) and combined in barcoded, taredvials. The resulting solutions were sampled for LC/MS analysis,concentrated in vacuo and their masses and yields were determined astabulated in the Tables.

If no additional purification was carried out after completion of theParallel Synthesis, this is indicated as “None” in Table 2.

Analytical HPLC Procedures. Analytical HPLC procedures were carried outaccording in one of two specific methods, depending upon availability ofinstrumentation and sample requirements, as follows.

HPLC Method A. Column: Thomson Inst. Co. 4.6×50 mm C18 5 μm 60 A; MobilePhase A: H₂O with 0.1% TFA; Mobile Phase B: CH₃CN with 0.1% TFA;Detection: UV 254 nm. Gradient 1: ELSD12MG; 10-90% B in 10 min, hold 90%B for 5 min; Flow: 1.0 mL/min. Gradient 2: ELSD5MG; 15-100% B in 5 min,hold 100% B for 3 min; Flow—2.0 mL/min.

HPLC Method B. Column: Thomson Inst. Co. 21×50 mm C18 5 μm 60 A; MobilePhase A: H₂O with 0.1% TFA; Mobile Phase B: CH₃CN with 0.1% TFA;Detection: UV 254 nm. Gradient 1: MIC8MG; 0-100% B in 8 min, hold 100% Bfor 2 min; Flow: 0.5 mL/min. Gradient 2: MIC15MG; 10-90% B in 15 min,hold 90% B for 3 min; Flow: 0.5 mL/min.

Preparative HPLC Procedures. Preparative HPLC was carried as follows.Instrument: Gilson; Column: Thomson Inst. Co. 21.5×150 mm C18 5 μm 60 A;Mobile Phase A: H₂O; Mobile Phase B: CH₃CN; Gradient: 15-100% B in 10min, hold 100% B for 5 min; Flow rate: 22 mL/min; Detection: UV 254 mm.The fractions containing the desired compounds were collected inbarcoded, tared vials, sampled for LC/MS analysis, concentrated in vacuoand their masses and yields were determined as shown in the Tables.

Spectroscopic and Other Instrumental Procedures.

NMR. The ¹H and ¹³C NMR spectra described herein were obtained usingVarian INOVA600 (600 MHz), Varian UNITY600 (600 MHz), or Varian 400 (400MHz) spectrometers. Spectrometer field strength and NMR solvent used fora particular sample are indicated in the Examples, or on any NMR spectraactually shown as Figures. Typically, ¹H NMR chemical shifts arereported as δ values in parts per million (ppm) downfield fromtetramethylsilane (TMS) (δ=0 ppm) as an internal standard, and ¹³C NMRchemical shifts are reported in ppm downfield from TMS and referencedwith respect to the CDCl₃ signal center line (δ=77.0 ppm). Solid orliquid samples were dissolved in an appropriate NMR solvent (CDCl₃ orDMSO-d₆), placed in a NMR sample tube, and data were collected accordingto the spectrometer instructional manuals. Most samples were analyzed inVariable Temperature mode, typically at about 55° C., though some datafor some samples were collected with the probe at ambient temperature.NMR data were processed using NUTS: NMR Utility Transform Software (LiteVersion-20011128) by Acorn NMR.

LC-MS. The Liquid Chromatography-Mass Spectrometry (LC-MS)instrumentation used to examine the compounds of the present inventionwas typically a quadrupole/time-of-flight mass spectrometer, withelectrospray ionization (ESI). For example, the typical LC-MSinstrumentation used was a Micromass Q-Tof using electrospray ionization(ESI). This instrument is a quadrupole/time-of-flight mass spectrometercapable of mass resolution up to m/z of about 7500. Samples wereintroduced in a direct injection mode by first dissolving and dilutingthe sample in methanol or acetonitrile and injecting the sample solutioninto the ESI source via a 10 μL loop Rheodyne injection valve. Thecarrier solvent was typically a mixture of 70% CH₃CN or MeOH and 30% H₂O(v:v), containing about 0.1% formic acid. Accurate mass analyses wereperformed in a similar fashion except for using a multipoint masscalibration with the same instrument under high mass resolutionconditions. Samples were spiked with an appropriate internal massreference compound, as known by one of ordinary skill, and analyzed asdescribed above.

Example 2 General Methods for Parallel Synthesis

Examples 3-5 describe the synthetic procedures for the preparation ofthe “library” of N²,N⁴,N⁶-tris(amino)-1,3,5-triazines which was preparedbased on the strategy of changing only one pendant amino group persynthesis, and based on the parent structure 95 shown below, where eachcompound in the library contains two of the pendant groups in 95.

The library was divided into three subgroups, and all three subgroupsare presented in Table 2. Library I (compounds 1-50) includes compoundshaving unchanged cycloheptylamino and[(1-ethyl-2-pyrrolidinyl)methyl]amino substituents, with various groupsbeing permuted at the remaining triazine amino position, prepared byMethod A as presented in Example 3. Library II (compounds 51-75)includes compounds having unchanged[(1-ethyl-2-pyrrolidinyl)methyl]amino and(3-fluoro-4-methoxyphenyl)amino substituents, with various groups beingpermuted at the remaining triazine amino position, prepared by Method Bas presented in Example 4. Library III (Compounds 76-100) includescompounds having unchanged (3-fluoro-4-methoxyphenyl)amino andcycloheptylamino substituents, with various groups being permuted at theremaining triazine amino position, prepared by Method C as described inExample 5. Thus, the combination of the specific amines employedproduced a library of compounds of novel composition. The sequence inwhich each monomer is added to form the compounds of the library is alsopresented in Table 2, because the Monomer 1 amine is added first,Monomer 2 amine added second, and Monomer 3 amine is added third.

Example 3 Parallel Synthetic Method A, for Library I Compounds

The following reaction scheme presents the general reagents andconditions for parallel synthetic method A used for the compounds ofTable 2 which designate Method A.

A stock solution of cyanuric chloride (0.542 M) in 1,4-dioxane wasprepared and 1 mL of this solution (containing 100 mg or 0.542 mmol) wasdispensed into each of 50 barcoded 40 mL vials. These solutions werecooled to about −11° C. (freezing) using a J-KEM block connected to acirculating cooler. Meanwhile, individual solutions of each aryl amineArNHR (specified as Monomer 1 in Table 2, 0.542 mmol) anddisopropylethylamine (DIEA) (77 mg/104 μL, 0.596 mmol) in 1 mL of CH₃CNwere prepared. (For HCl salts, 204 μL DIEA (approx. 2.1 equiv) wasused.) Over a period of about 1 h, the amine/DIEA solutions were addedto the corresponding frozen cyanuric chloride solutions, one by one,with swirling. The resulting solutions were then shaken at about −11° C.for about 1 h and the reaction block was allowed to warm to roomtemperature over the next hour. The resulting2-amino-4,6-dichlorotriazine solutions were carried to the next stepwithout purification.

A stock solution of cycloheptylamine (1.08 M) and DIEA (1.19 M) in CH₃CNwas prepared and 0.5 mL (containing 61 mg/69 μL, 0.542 mmol amine and 77mg/104 μL, 0.596 mmol DIEA) was dispensed into each of the 40 mL vialsfrom the first step. The vials were shaken on the J-KEM block overnightat room temperature and placed in a freezer (about −14° C.) withoutpurification until the next reaction.

A stock solution of 2-(aminomethyl)-1-ethylpyrrolidine (1.08 M) and DIEA(1.19 M) in CH₃CN was prepared and 0.5 mL (containing 69 mg/79 μL, 0.542mmol amine and 77 mg/104 μL, 0.596 mmol DIEA) was dispensed into each ofthe 40 mL from the second step. The vials were then shaken on the J-KEMblock at about 80° C. for about 15 h. The solutions were cooled to roomtemperature and taken to dryness in vacuo. The residues were thenextracted with ethyl acetate and the extract was washed with brine. Theaqueous layers were extracted a second time with ethyl acetate and thecombined organic layers were dried over Na₂SO₄ and passed through a plugof Celite™ into barcoded, tared vials. After concentration in vacuo,masses were determined and yields were calculated, and the compoundswere sampled for LC/MS analysis.

Example 4 Parallel Synthetic Method B, for Library II Compounds

The following reaction scheme presents the general reagents andconditions for parallel synthetic method B, used for the compounds ofTable 2 which designate Method B.

In an oven-dried round bottom flask, a solution of cyanuric chloride(5.0 g, 27.1 mmol) in 1,4-dioxane (40 mL) was cooled to freezing in aCH₃CN/dry ice bath. To this frozen solution was added 40 mL of CH₃CN,followed by DIEA (3.85 g/5.19 mL, 29.8 mmol). A solution of3-fluoro-p-anisidine (3.83 g, 27.1 mmol) in 10 mL of CH₃CN was thenadded slowly via syringe. The reaction mixture was stirred at about −20°C. for about 1 h and allowed to warm to room temperature over about 1 h.The resulting 2-amino-4,6-dichlorotriazine solution was carried to thenext step without purification.

Fifty mL (13.5 mmol) of the prepared(4,6-dichloro-[1,3,5]triazin-2-yl)-(3-fluoro-4-methoxy-phenyl) aminesolution was divided equally (2 mL or 0.54 mmol each) among 25 barcoded,40 mL scintillation vials. Individual solutions of each R₂NHR (where R₂amine indicates Monomer 2 in Table 2, 0.542 mmol) and DIEA (77 mg/104μL, 0.596 mmol) in 0.5 mL of CH₃CN were prepared and added to thecorrespondingly labeled 40 mL vials. The resulting solutions were shakenon the J-KEM block overnight at room temperature and then placed in afreezer (about −14° C.) without purification until the next reaction.

A stock solution of 2-(aminomethyl)-1-ethylpyrrolidine (1.08 M) and DIEA(1.19 M) in CH₃CN was prepared and 0.5 mL (containing 69 mg/79 μL, 0.542mmol amine and 77 mg/104 μL, 0.596 mmol DIEA) was dispensed into each ofthe 40 mL vials from the second step. The vials were shaken on the J-KEMblock at about 80° C. for about 15 h. The solutions were cooled to roomtemperature and concentrated in vacuo. The residues were then extractedwith ethyl acetate and the extract washed with brine. The aqueous layerswere extracted a second time with ethyl acetate and the combined organiclayers were dried over Na₂SO₄ and passed through a plug of Celite™ intobarcoded, tared vials. After concentration in vacuo, masses werecalculated and the compounds were sampled for LC/MS analysis.

Example 5 Parallel Synthetic Method C, for Library III Compounds

The following reaction scheme presents the general reagents andconditions for parallel synthetic method C, used for the compounds ofTable 2 which designate Method C.

In an oven-dried round bottom flask, a solution of cyanuric chloride(5.0 g, 27.1 mmol) in 1,4-dioxane (40 mL) was cooled to freezing in aCH₃CN/dry ice bath. To this frozen solution was added 40 mL of CH₃CN,followed by DIEA (3.85 g/5.19 mL, 29.8 mmol). A solution of3-fluoro-p-anisidine (3.83 g, 27.1 mmol) in 10 mL of CH₃CN was thenadded slowly via syringe. The reaction mixture was stirred at about −20°C. for about 1 h and allowed to warm to room temperature over 1 h. Theresulting 2-amino-4,6-dichlorotriazine solution was carried to the nextstep without purification.

Fifty mL (13.5 mmol) of the prepared(4,6-dichloro-[1,3,5]triazin-2-yl)-(3-fluoro-4-methoxy-phenyl) aminesolution was treated with a solution of cycloheptylamine (1.53 g/1.73mL, 13.5 mmol) and DIEA (1.93 g/2.60 mL, 14.9 mmol) in CH₃CN (8 mL). Theresulting solution was stirred overnight at room temperature and carriedto the next step without purification.

The resulting6-chloro-N-cycloheptyl-N-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diaminesolution (13.5 mmol) was diluted up to 62.5 mL with CH₃CN and dividedequally (2.5 mL or 0.54 mmol each) between 25 barcoded 40 mLscintillation vials. Individual solutions of each R₃NHR (where R₃ amineindicates Monomer 3 in Table 2, 0.542 mmol) and DIEA (77 mg/104 μL,0.596 mmol) in 0.5 mL of CH₃CN were prepared and added to thecorrespondingly labeled 40 mL vial. The resulting solutions were shakenon the J-KEM block at about 80° C. for about 15 h. The solutions werecooled to room temperature and concentrated in vacuo. The residues werethen extracted with ethyl acetate and the extract washed with brine.Each organic layer was dried over Na₂SO₄ and passed through a plug ofCelite™ into a barcoded, tared vial. After concentration in vacuo,masses were calculated and the compounds were sampled for LC/MSanalysis.

Example 6 Synthesis of6-Chloro-N-(3-chloro-4-methoxy-phenyl)-N′-cyclohexylmethyl-[1,3,5]triazine-2,4-diamine(102)

To a sample of 101 (0.3004 g, 1.0 mmol, prepared as indicated herein)dissolved in acetone (4 mL) was added a solution ofcyclohexanemethylamine (0.13 mL, 1.0 mmol) in acetone (1 mL) followed byaddition of a NaOH solution (0.0448 g, 1.0 mmol dissolved in 1 mL ofH₂O). The reaction mixture was allowed to stir at reflux for about 3hours. The reaction mixture was then poured over crushed ice andneutralized with 10% HCl (aq) and 5% NaOH (aq). The resulting solid wascollected by vacuum filtration, washed with water and dried overnightunder vacuum to afford compound 102 (0.29 g, 76% recovery).

Example 7 Synthesis ofN-(3-Chloro-4-methoxy-phenyl)-N′-cyclohexylmethyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine(103)

To a sample of 102 (0.286 g, 1.0 mmol) dissolved in 1,4-dioxane (4 mL)was added a solution of N-methyl-4(methylamino)piperidine (0.15 mL, 1.0mmol) in acetone (1 mL) followed by addition of a NaOH solution (0.0462g, 1.0 mmol dissolved in 1 mL of H₂O). The reaction mixture was allowedto stir at about 80° C. for about 2 hours. The reaction mixture waspoured over crushed ice and neutralized with 10% HCl (aq). The resultingsolid was collected by vacuum filtration, washed with water and driedunder, vacuum overnight. Column chromatography (silica gel, 96:3:1dichloromethane:methanol:conc. ammonium hydroxide) yielded a lightpurple solid 103 (41 mg, 9%), mp 84° C.; HPLC: YMC Pack Pro C18,40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 12.7 min,97% purity); ¹H NMR (600 MHz, CDCl₃, 55° C.) δ 7.98 (s, 1H), 7.18 (S,1H), 6.85 (d, J=9 Hz, 1H), 6.58 (s, 1H), 4.89 (s, 1H), 4.58-4.62 (m,1H), 3.87 (s, 3H), 3.25 (t, J=6.6 Hz, 2H), 3.05 (s, 3H), 2.94 (d, J=11.4Hz, 2H), 2.31 (s, 3H), 2.15 (S, 2H), 1.86 (dq, J=12, 4.2 Hz, 3H),1.57-1.78 (m, 8H), 1.15-1.30 (m, 4H), 1.00 (dq, J=11.4, 3 Hz, 2H); MS(ESI): m/z 476 (37.7), 474 (M+H, 100), 410 (1.4).

Example 8 Synthesis of6-Chloro-N-(3-chloro-4-methoxy-phenyl)-N′-(1-propyl-butyl)-[1,3,5]triazine-2,4-diamine(104)

To a sample of 101 (0.3062 g, 1.0 mmol) dissolved in acetone (4 mL) wasadded a solution of 4-heptylamine (0.15 mL, 1.0 mmol) in acetone (1 mL)followed by addition of a NaOH solution (0.0410 g, 1 mmol dissolved in 1mL of H₂O). The reaction mixture was allowed to stir at 30-50° C. forabout about 3 hours. The reaction mixture was then poured over crushedice and neutralized with 10% HCl (aq) and 5% NaOH (aq). The resultingsolid was collected by vacuum filtration, washed with water and driedovernight under vacuum to afford 104 (0.363 g, 94% recovery).

Example 9 Synthesis ofN-(3-Chloro-4-methoxy-phenyl)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-(1-propyl-butyl)-[1,3,5]triazine-2,4,6-triamine(105)

To a sample of 104 (0.363 g, 1.0 mmol) dissolved in 1,4-dioxane (6 mL)was added a solution of N-methyl-4(methylamino)piperidine (0.15 mL, 1.0mmol) in acetone (1 mL) followed by addition of a NaOH solution (0.0414g, 1.0 mmol dissolved in 1 mL of H₂O). The reaction mixture was allowedto stir at about 80° C. for about about 2 hours. The reaction mixturewas poured over crushed ice and neutralized with 10% HCl (aq). Theresulting solid was collected by vacuum filtration, washed with waterand dried under vacuum overnight. Column chromatography (silica gel,96:3:1 dichloromethane:methanol:conc. ammonium hydroxide) yielded lightpurple solid 105 (97 mg, 20%), mp 249° C. HPLC: YMC Pack Pro C18,40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 14.4 min,98% purity; MS (ESI): m/z 476 (M+H, 100), 412 (2.9), 366 (2.8), 239(1.9).

Example 10 Synthesis ofN-(3-Chloro-4-methoxy-phenyl)-N′-isopropyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine(106)

To a sample of 101 (0.6157 g, 2.0 mmol) dissolved in anhydrous1,4-dioxane (15 mL) was added a solution of isopropylamine (0.17 mL, 2.0mmol) in anhydrous acetonitrile (1 mL) followed by addition of aN,N-diisopropylethylamine (DIEA) (0.38 mL, 2.2 mmol) in anhydrousacetonitrile (1 mL). The reaction mixture was allowed to stir at roomtemperature overnight under nitrogen. To this mixture was added DIEA(0.38 mL, 2.2 mmol) in anhydrous acetonitrile (1 mL) followed byaddition of N-methyl-4(methylamino)piperidine (0.29 mL, 2.0 mmol) inanhydrous acetonitrile (1 mL). The reaction mixture was allowed to stirat reflux overnight under nitrogen. The reaction mixture was extracted 3times with ethyl acetate. The combined organic layers were washed onetime with brine solution and dried over anhydrous potassium carbonate.The organic layer was concentrated on a rotary evaporator and allowed todry overnight under vacuum. Column chromatography (silica gel, 93:6:1CH₂Cl₂:CH₃OH:conc. NH₄OH) yielded light brown solid 106 (271 mg, 32%);TLC (silica gel, 93:6:1 CH₂Cl₂:CH₃OH:conc. NH₄OH), R_(f) 0.28; HPLC:Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264nm, R_(t) 4.4 min, 84.8% purity; MS (ESI): m/z 422 (26), 420 (M+H,71.2), 378 (4.2), 231 (100), 211 (40.4), 118 (5.4).

Example 11 Synthesis ofN²-(3-chloro-4-methoxy-phenyl)-N⁴-isopropyl-N⁶-methyl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine(107)

Compound 107 was isolated (0.159 g) as a by-product via columnchromatography (silica gel, 93:6:1 CH₂Cl₂:CH₃OH:conc. NH₄OH); mp 129°C.; TLC (silica gel, 93:6:1 CH₂Cl₂:CH₃OH:conc. NH₄OH), R_(f) 0.14; HPLC:Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264nm, R_(t) 4.4 min, 93.5% purity; MS (ESI): m/z 408 (17.2), 406 (M+H,46.6), 375 (18.5), 245 (11.9), 224 (100), 204 (13.4).

Example 12 Synthesis of5-{4-(3-Chloro-4-methoxy-phenylamino)-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-pentan-1-ol(108)

To a sample of 101 (1.5046 g, 5.0 mmol) dissolved in anhydrous1,4-dioxane (30 mL) was added a solution of 5-amino-1-pentanol (0.5067g, 5.0 mmol) in anhydrous acetonitrile (12 mL) followed by addition ofN,N-diisopropylethylamine (DIEA) (0.95 mL, 5.5 mmol) in anhydrousacetonitrile (2 mL). The reaction mixture was allowed to stir at roomtemperature overnight under nitrogen. To the reaction mixture was addedDIEA (0.95 mL, 5.5 mmol) in anhydrous acetonitrile (1 mL) followed byaddition of N-methyl-4(methylamino)piperidine (0.73 mL, 5.0 mmol) inanhydrous acetonitrile (1 mL). The reaction mixture was allowed to stirat reflux overnight under nitrogen. The reaction mixture was extracted 3times with ethyl acetate. The combined organic layers were washed onetime with brine solution and dried over anhydrous potassium carbonate.The organic layer was concentrated on a rotary evaporator and allowed todry overnight under vacuum. Column chromatography (silica gel, 90:9:1CH₂Cl₂:CH₃OH:conc. NH₄OH) yielded light brown solid 108 (300 mg, 13%);TLC (silica gel, 90:9:1 CH₂Cl₂:CH₃OH:conc. NH₄OH), R_(f) 0.22; HPLC: YMCPack Pro C18, 40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm,R_(t) 3.5 min, 74.8% purity; MS (ESI): m/z 466 (24.2), 464 (M+H, 71.5),378 (5.2), 253 (4.5), 244 (20.5), 233 (100), 216 (33.3), 196 (14.6), 118(5.1).

Example 13 Synthesis of5-[4-(3-chloro-4-methoxy-phenylamino)-6-(methyl-piperidin-4-yl-amino)-1,3,5-triazin-2-ylamino]-pentan-1-ol(109)

Compound 109 was isolated as a by-product (0.820 g) via columnchromatography (silica gel, 90:9:1 CH₂Cl₂:CH₃OH:conc. NH₄OH), mp 101°C.; TLC (silica gel, 90:9:1 CH₂Cl₂:CH₃OH:conc. NH₄OH), R_(f) 0.08; HPLC:Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264nm, R_(t) 3.6 min, 95.3% purity; MS (ESI): m/z 452 (13), 450 (M+H,35.6), 419 (3.9), 267 (5.1), 246 (100), 226 (21.3), 209 (23.6), 118(1.1).

Example 14 Synthesis ofN-Butyl-6-chloro-N′-(3-chloro-4-methoxy-phenyl)-N-propyl-[1,3,5]triazine-2,4-diamine(110)

To a sample of 101 (1.5334 g, 5.0 mmol) dissolved in acetone (20 mL) wasadded a solution of N-propyl-butylamine (0.77 mL, 5.0 mmol) in acetone(1 mL) followed by the addition of NaOH (2.0 mL, 2.5 N, 5.0 mmol). Thereaction mixture was allowed to stir at 30-35° C. for about 3 hoursunder nitrogen. The reaction mixture was extracted 3 times withdichloromethane; the combined organic layers were washed with brinesolution and dried over potassium carbonate. The sample was concentratedon a rotary evaporator and the resulting oil was dried overnight undervacuum. Column chromatography (silica gel, 96:3:1dichloromethane:methanol:conc. ammonium hydroxide) yielded light brownsolid 110 (1.4 g, 77% recovery).

Example 15 Synthesis ofN-Butyl-N′-(3-chloro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-N-propyl-[1,3,5]triazine-2,4,6-triamine(111)

To a sample of 110 (1.323 g, 3.4 mmol) dissolved in 1,4-dioxane (25 mL)was added a solution of N-methyl-4(methylamino)piperidine (0.4 mL, 3.4mmol) in 1,4-dioxane (1 mL) followed by the addition of NaOH (1.4 mL,2.5 N, 3.4 mmol). The reaction mixture was allowed to stir at reflux forabout 2 hours under nitrogen. The reaction mixture was extracted 3 timeswith dichloromethane; the combined organic layers were washed with brineand dried over potassium carbonate. The sample was concentrated on therotary evaporator and the resulting solid was dried overnight undervacuum. Column chromatography (silica gel, 90:9:1dichloromethane:methanol:conc. ammonium hydroxide) yielded light brownsolid compound 111 (527 mg, 33%), mp 68° C.; TLC (silica gel, 90:9:1CH₂Cl₂:CH₃OH:conc. NH₄OH), R_(f) 0.46; HPLC: ODS-3V C18, 40:30:30[KH₂PO₄ (0.0M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 41.6 min, 90.8%purity); MS (ESI): m/z 476 (M+H, 28.5), 261 (20.2), 260 (52.8), 259(100), 239 (18.6), 239 (50.6).

Example 16 Synthesis ofN²-Butyl-N⁴-(3-chloro-4-methoxy-phenyl)-N⁶-methyl-N⁶-piperidin-4-yl-N²-propyl-1,3,5-triazine-2,4,6-triamine(112)

Compound 112 was isolated as a by-product via column chromatography, anoil (0.112 g); TLC (silica gel, 90:9:1 CH₂Cl₂:CH₃OH:conc. NH₄OH), R_(f)0.23; HPLC: ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN],265 nm, R_(t) 41.4 min, 97.8% purity); MS (ESI): m/z 464 (11.6), 462(M+H, 28.9), 431 (15.6), 273 (12.7), 253 (58.8), 252 (100), 232 (25.8),157 (14.5).

Example 17 Synthesis of 2,4-Dichloro-6-cyclohexylmethoxy-[1,3,5]triazine(113)

To cyanuric chloride (3.76 g, 20.0 mmol) dissolved in toluene (20 mL)was added potassium bicarbonate (2.80 g, 20.0 mmol) and 18-crown-6(0.1614 g, 0.6 mmol) followed by dropwise addition of cyclohexylmethanol(2.5 mL, 20 mmol) in 15 mL of toluene (15 mL). The reaction mixture wasallowed to stir at reflux for about 18 hours under nitrogen. Thereaction mixture was passed through a plug of Celite and concentratedusing a rotary evaporator and dried over night under vacuum to give 113as an oil (5.212 g, 99% recovery).

Example 18 Synthesis of(4-Chloro-6-cyclohexylmethoxy-[1,3,5]triazin-2-yl)-(3-fluoro-4-methoxy-phenyl)-amine(114)

To a sample of 113 (1.011 g, 3.8 mmol) dissolved in acetone (20 mL) wasadded a solution of 3-fluoro-p-anisidine (0.541 g, 3.8 mmol) in acetone(10 mL) followed by addition of NaOH (1.52 mL, 2.5 N, 3.8 mmol) andwater (3 mL). The reaction mixture was allowed to stir at reflux forabout 3 hours under nitrogen. The reaction mixture was extracted 3 timeswith dichloromethane; the combined organic layers were washed with brinesolution and dried over potassium carbonate. The sample was concentratedon a rotary evaporator and the resulting oil was dried overnight undervacuum. Column chromatography (silica gel, 70:30 hexanes:ethyl acetate)yielded light yellow solid compound 114 (0.581 g, 42%), mp 98° C.; TLC(silica gel, 30:70 ethyl acetate:hexanes), R_(f) 0.36; MS (ESI): m/z 369(39.1), 368 (22.1), 367 (M+H, 100), 273 (3.2), 271 (10.7).

Example 19 Synthesis of6-Cyclohexylmethoxy-N,N′-bis-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4-diamine(115)

Compound 115 was obtained as a by-product (0.159 g) via columnchromatography (silica gel, 70:30 hexanes:ethyl acetate), mp 181° C.;TLC (silica gel, 30:70 ethyl acetate:hexanes), R_(f) 0.17; MS (ESI): m/z472 (M+H, 100), 261 (1.5).

Example 20 Synthesis of6-Cyclohexylmethoxy-N-(1-ethyl-pyrrolidin-2-ylmethyl)-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine(116)

To a sample of 114 (0.3004 g, 0.82 mmol) dissolved in 1,4-dioxane (15mL) was added a solution of 2-(aminomethyl)-1-ethylpyrrolidine (0.12 mL,0.82 mmol) in acetone (1 mL) followed by the addition of NaOH (0.33 mL,2.5 N, 0.82 mmol) and water (1 mL). The reaction mixture was allowed tostir at reflux for about 2 hours under nitrogen. The reaction mixturewas extracted 3 times with dichloromethane; the combined organic layerswere washed with brine and dried over potassium carbonate. The samplewas concentrated on the rotary evaporator and the resulting solid wasdried overnight under vacuum. Column chromatography (silica gel, 93:6:1dichloromethane:methanol:conc. ammonium hydroxide) yielded a lightyellow solid, compound 116 (226 mg, 60%), mp 59° C.; HPLC: InertsilODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t)10.5 min, 100% purity; ¹H NMR (600 MHz, CDCl₃, 55° C.) δ 7.65 (broadresonance, rotamers, 1H), 7.07 (br d, J=7.8 Hz, 1H), 6.90 (t, J=9 Hz,1H) 6.84 (broad resonance, rotamers, 1H), 4.12 (s, 2H), 3.88 (S, 3H),1.02 (s, 1H), 2.26 (apt sextet, J=6.6 Hz, 1H), 2.19 (q, J=9 Hz, 1H),1.16-1.92 (m, 10H), 1.57 (s, 2H), 1.17-1.32 (m, 3H), 1.05-1.11 (m, 4H);MS (ESI): m/z 459 (M+H, 100), 363 (40.7), 223 (16.1), 202 (4.4), 138(1.2).

Example 21 Synthesis of(4-Chloro-6-cyclohexylmethoxy-[1,3,5]triazin-2-yl)-(3-chloro-4-methoxy-phenyl)-amine(117)

To a sample of compound 113 (1.012 g, 3.8 mmol) dissolved in acetone (20mL) was added a solution of 3-chloro-p-anisidine (0.605 g, 3.8 mmol) inacetone (10 mL) followed by addition of NaOH (1.52 mL, 2.5 N, 3.8 mmol)and water (3 mL). The reaction mixture was allowed to stir at reflux forabout 3 hours under nitrogen. The reaction mixture was extracted 3 timeswith dichloromethane; the combined organic layers were washed with brineand dried over potassium carbonate. The sample was concentrated on arotary evaporator and the resulting oil was dried overnight undervacuum. Column chromatography (silica gel, 70:30 hexanes:ethyl acetate)yielded a light peach colored solid, compound 117 (0.547 g, 38%), mp114° C.; TLC (silica gel, 30:70 ethyl acetate:hexanes), R_(f) 0.44; MS(ESI): m/z 385 (74.3), 384, (22.9), 383 (M+H, 100), 287 (8.3).

Example 22 Synthesis ofN,N′-Bis-(3-chloro-4-methoxy-phenyl)-6-cyclohexylmethoxy-1,3,5-triazine-2,4-diamine(118)

Compound 118 was obtained as a by-product (0.178 g) via columnchromatography (silica gel, 70:30 hexanes:ethyl acetate), mp 188° C.;TLC (silica gel, 30:70 ethyl acetate:hexanes), R_(f) 0.22; MS (ESI): m/z504 (M+H, 100), 379 (1), 338 (1.3).

Example 23 Synthesis ofN-(3-Chloro-4-methoxy-phenyl)-6-cyclohexylmethoxy-N′-methyl-N′-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4-diamine(119)

To a sample of 117 (0.3007 g, 0.78 mmol) dissolved in 1,4-dioxane (15mL) was added a solution of 2-(aminomethyl)-1-ethylpyrrolidine (0.11 mL,0.78 mmol) in acetone (1 mL) followed by the addition of NaOH (0.31 mL,2.5 N, 0.78 mmol) and water (1 mL). The reaction mixture was allowed tostir at reflux for about 2 hours under nitrogen. The reaction mixturewas extracted 3 times with dichloromethane; the combined organic layerswere washed with brine solution and dried over potassium carbonate. Thesample was concentrated on the rotary evaporator and the resulting solidwas dried overnight under vacuum. Column chromatography (silica gel,93:6:1 dichloromethane:methanol:conc. ammonium hydroxide) yielded lightyellow solid compound 119 (159 mg, 43%), mp 140° C. HPLC: InertsilODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t)15.2 min, 99.7% purity; MS (ESI): m/z 475 (M+H, 64.1), 379 (49.5), 231(48.6), 210 (100), 190 (3.2).

Example 24 Synthesis of6-Chloro-N,N″-bis-(3-chloro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine(120)

To a sample of 101 (3.0556 g, 10.0 mmol) dissolved in acetone (25 mL)was added a solution of 3-chloro-p-anisidine (1.6050 g, 10.0 mmol) inacetone (10 mL) followed by addition of NaOH (4.0 mL, 2.5 N, 10.0 mmol).The reaction mixture was allowed to stir at rt for about 3 hours undernitrogen. The reaction mixture was poured over crushed ice. Theresulting solid was collected by vacuum filtration, washed with waterand dried overnight under vacuum to give compound 120 (4.06 g, 95%), mp213° C.; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 70.0 min, 97.1% purity MS (ESI): m/z427 (20.90), 426 (M+H, 99.6), 210 (100), 209 (22.2), 196 (55.3), 169(25.4).

Example 25 Synthesis ofN,N′-Bis-(3-chloro-4-methoxy-phenyl)-N″-methyl-N″-(4-methyl-cyclohexyl)-[1,3,5]triazine-2,4,6-triamine(121)

To a sample of compound 120 (1.5004 g, 3.5 mmol) dissolved in1,4-dioxane (20 mL) was added a solution ofN-methyl-4(methylamino)-piperidine (0.5 mL, 3.5 mmol) in 1,4-dioxane (1mL) followed by the addition of NaOH (1.4 mL, 2.5 N, 3.5 mmol). Thereaction mixture was allowed to stir at reflux for about 2 hours undernitrogen. The reaction mixture was poured over crushed ice andneutralized with 10% HCl (aq). The resulting solid was collected byvacuum filtration, washed with water and dried overnight under vacuum.Column chromatography (silica gel, 96:3:1 dichloromethane:methanol:conc.ammonium hydroxide) yielded a purple solid, compound 121 (487 mg, 27%),mp 130° C.; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 8.1 min, 96% purity; ¹H NMR (600 MHz,CDCl₃, 55° C.) δ 7.81-7.92 (broad resonance, 2H), 7.19-7.30 (broadresonance, 2H), 6.87 (d, J=9 Hz, 2H), 6.72 (s, 2H), 4.60-4.65 (m, 1H),3.88 (s, 6H), 3.05 (s, 3H), 2.95 (d, J=12 Hz, 2H), 2.32 (s, 3H), 2.19(t, J=11.4 Hz, 2H), 1.89 (dq, J=12.6, 3.6 Hz, 2H), 1.71 (apt d, J=11.4Hz, 2H), 1.65 (s, 1H); MS (ESI): m/z 519 (28.3), 518 (M+H, 42.1), 261(71.9), 260 (100).

Example 26 Synthesis ofN,N′-Bis-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-triamine(122)

To a sample of 120 (1.5004 g, 3.5 mmol) dissolved in acetone (20 mL) wasadded a solution of cycloheptylamine (0.4 mL, 3.5 mmol) in acetone (1mL) followed by the addition of NaOH (1.4 mL, 2.5 N, 3.5 mmol). Thereaction mixture was allowed to stir at reflux for about 2 hours undernitrogen. The reaction mixture was poured over crushed ice andneutralized with 10% HCl (aq). The resulting solid was collected byvacuum filtration, washed with water and dried overnight under vacuum togive light purple solid compound 122 (1.5 g, 85%), mp 183° C.; HPLC:Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264nm, R_(t) 59 min, 96% purity; MS (ESI): m/z 503 (M+H, 29), 502 (100),458 (24.2), 425 (17.9), 225 (5.7), 155 (11.3), 114 (27.6).

Example 27 Synthesis ofN-(3-Bromo-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine(123)

To cyanuric chloride (0.184 g, 1.0 mmol) dissolved in acetonitrile (3mL) stirring at about −10° C., was added a solution of3-bromo-p-anisidine (0.2019 g, 1.0 mmol) in acetonitrile followed by theaddition of N,N-diisopropylethylamine (DIEA) (0.17 mL, 1.0 mmol) inacetonitrile. The reaction mixture was allowed to stir at about −10° C.for 1 hour under nitrogen. The reaction mixture was then warmed to roomtemperature and allowed to stir at room temperature for another hourunder nitrogen. To the reaction mixture was added a solution ofcycloheptylamine (0.13 mL, 1.0 mmol) in acetonitrile followed byaddition of DIEA (0.17 mL, 1.0 mmol). The reaction mixture was allowedto stir at reflux overnight under nitrogen. To the reaction mixture wasadded N-methyl-4(methylamino)piperidine (0.13 mL, 1.0 mmol) inacetonitrile followed by the addition of DIEA (0.17 mL, 1.0 mmol). Thereaction mixture was allowed to stir at reflux for overnight undernitrogen. The reaction mixture was extracted 3 times with ethyl acetate;the combined organic layers were washed with brine solution and driedover potassium carbonate. The sample was concentrated on the rotaryevaporator and the resulting solid was dried overnight under vacuum.Column chromatography (silica gel, 90:9:1 methylenechloride:methanol:conc. ammonium hydroxide yielded 0.029 g (6%) of 123.¹H NMR (400 MHz, CDCl₃) δ 7.97-8.19 (broad resonance, 1H), 7.12 (broadresonance, 1H), 6.78-6.80(m, 2H), 4.82 (br s, 1H), 4.58 (br s, 1H), 3.92(br s, 1H), 3.84 (s, 3H), 2.90-2.98 (m, 5H), 2.29 (s, 3H), 2.17 (broadresonance, 2H), 1.99-2.24 (broad resonance, 4H), 1.72-1.85 (m, 3H),1.42-1.62 (m, 11H); MS (ESI): m/z 520 (100), 518 (93.9), 458 (10.4), 424(20.8), 422 (21.1), 261 (67.5), 260 (63.4), 213 (13.9), 212 (13.6).

Example 28 Synthesis of6-Chloro-N-cyclohexylmethyl-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine(125)

To a sample of 124 (40.02 g, 138.4 mmol, prepared as indicated herein)dissolved in acetone (300 mL) was added a solution ofcyclohexanemethylamine (18.0 mL, 138.4 mmol) in acetone (30 mL) followedby addition of NaOH (55.4 mL, 2.5 N, 138.4 mmol) and 130 mL of water.The reaction mixture was allowed to stir at reflux for about 3 hours.The reaction mixture was then poured over crushed ice and neutralizedwith 10% HCl (aq) and 10% NaOH (aq). The resulting solid was collectedby vacuum filtration, washed with water and dried overnight undervacuum. Recrystallization from ethyl acetate yielded a light yellowsolid, compound 125 (32.93 g, 65%), mp 156° C.; HPLC: Inertsil ODS-3VC18, 40:10:50 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 47.9min, 92% purity; MS (ESI): m/z 366 (M+H, 100).

Example 29 Synthesis ofN-Cyclohexylmethyl-N′-(1-ethyl-pyrrolidin-2-ylmethyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine(126)

To a sample of 125 (10.02 g, 27.3 mmol) dissolved in 1,4-dioxane (150mL) was added a solution of 2-(aminomethyl)-1-ethylpyrrolidine (4.0 mL,27.3 mmol) in acetone (10 mL) followed by addition of NaOH (11 mL, 2.5N, 27.3 mmol) and 27 mL of water. The reaction mixture was allowed tostir at reflux for about 2 hours. The reaction mixture was extracted 3times with dichloromethane; the combined organic layers were washed withbrine and dried over potassium carbonate. The sample was concentrated onthe rotary evaporator and the resulting solid was dried overnight undervacuum. Column chromatography (silica gel, 93:6:1dichloromethane:methanol:conc. ammonium hydroxide) yielded a lightyellow solid, compound 126 (7.014 g, 56%), mp 72° C.; HPLC: InertsilODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t)8.5 min, 93.4% purity; MS (ESI): m/z 458 (M+H, 37.3), 362 (4), 250(100), 230 (15.3), 229 (44.1).

Example 30 Synthesis ofN-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-6-pyrrolidin-1-yl-[1,3,5]triazine-2,4-diamine(128)

To a sample of compound 127 (13.24 g, 36.2 mmol, prepared as indicatedherein) dissolved in THF (150 mL) was added a solution of pyrrolidine(3.0 mL, 36.2 mmol) in THF (10 mL) followed by addition of NaOH (14.5mL, 2.5 N, 36.2 mmol) and 36 mL of water. The reaction mixture wasallowed to stir at reflux for about 2.5 hours. The reaction mixture wasextracted 3 times with dichloromethane; the combined organic layers werewashed with brine and dried over potassium carbonate. The sample wasconcentrated on the rotary evaporator and the resulting solid was driedovernight under vacuum. Column chromatography (silica gel, 98:2dichloromethane:methanol) yielded light yellow solid 128 (3.36 g, 23%),mp 79° C.; HPLC: Inertsil ODS-3V C18, 40:10:50 [KH₂PO₄ (0.01M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 24.5 min, 95.5% purity; ¹H NMR (600MHz, CDCl₃, 55° C.) δ 7.77 (broad resonance, 1H), 7.01-7.03 (m, 1H),6.86 (t, J=9 Hz, 1 H), 6.62 (s, 1H), 4.80 (s, 1H), 4.02-4.06 (m, 1H),3.85 (s, 3H), 3.54 (s, 4 H), 1.99-2.03 (m, 2H), 1.91-1.93 (m, 3H),1.47-1.66 (m, 11H); MS (ESI): m/z 402 (30.7), 401 (M+H, 100).

Example 31 Synthesis of(4,6-Dichloro-[1,3,5]triazin-2-yl)-(3-fluoro-4-methoxy-phenyl)-amine(124)

To cyanuric chloride (28.84 g, 156.0 mmol) dissolved in acetone (200 mL)stirring at approximately 0-5° C., was added a solution of3-fluoro-p-anisidine (22.16 g, 156.0 mmol) in acetone (200 mL) followedby the addition of NaOH (63 mL, 2.5 N, 156.0 mmol). The reaction mixturewas allowed to stir at approximately 0-5° C. for about 2 hours. Thereaction mixture was then poured over crushed ice and neutralized with10% HCl (aq) and 5% NaOH (aq). The resulting solid was collected byvacuum filtration, washed with water and dried overnight under vacuum.Column chromatography (silica gel, 70:30 hexane:ethyl acetate) yieldedlight yellow solid compound 124 (29.6 g, 66%); mp 134° C.; HPLC:Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264nm, R_(t) 20.3 min, 97.7% purity.

Example 32 Synthesis of6-Chloro-N-cycloheheptyl-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine(127)

To a sample of 124 (10.00 g, 34.6 mmol) dissolved in acetone (150 mL)was added a solution of cycloheptylamine (4.4 mL, 34.6 mmol) in acetone(20 mL) followed by addition of NaOH (13.8 mL, 2.5 N, 34.6 mmol) and 35mL of water. The reaction mixture was allowed to stir at reflux forabout 3 hours. The reaction mixture was extracted 3 times withdichloromethane; the combined organic layers were washed with brine anddried over potassium carbonate. The sample was concentrated on therotary evaporator and the resulting solid was dried overnight undervacuum affording 127 (12.4 g, 98% recovery), mp 145° C.; HPLC: InertsilODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t)104.8 min, 97.3% purity; ¹H NMR (600 MHz, CDCl₃, 55° C.) δ 7.50-7.64 (m,1H), 7.02-7.03 (br resonance, 2H), 6.90 (t, J=8.9 Hz, 1H), 5.35-5.41 (brresonance, 1H), 3.99 (br s, 1H), 4.12 (rotamer), 3.87 (s, 3H), 2.01 (brs, 2H), 1.42-1.67 (m, 11H).

Example 33 Synthesis ofN-Cycloheptyl-N′-ethyl-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine(129)

To 127 (11.00 g, 30 mmol) dissolved in THF (150 mL) was added a solutionof ethylamine hydrochloride (2.43 mL, 30 mmol) in THF (20 mL) followedby addition of NaOH (24 mL, 2.5 N, 60 mmol) and 30 mL of water. Thereaction mixture was allowed to stir at reflux for about 2 hours. Thereaction mixture was extracted 3 times with dichloromethane; thecombined organic layers were washed with brine and dried over potassiumcarbonate. The sample was concentrated on the rotary evaporator and theresulting solid was dried overnight under vacuum. Column chromatography(silica gel, 98:2 dichloromethane:methanol) yielded a light yellow solid129 (4.81 g, 43%), mp 84° C.; HPLC: Inertsil ODS-3V C18, 40:30:30[KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 30.7 min, 94.2%purity; ¹H NMR (600 MHz, CDCl₃, 55° C.) δ 7.69 (s, 1H), 7.00 (br d,J=7.0 Hz, 1H), 6.86 (t, J=8.4 Hz, 1H), 6.64 (s, 1H), 4.79-4.83 (brresonance, 2H), 4.01-4.03 (m, 1H), 3.85 (s, 3H), 3.38-3.42 (m, 2H),1.99-2.01 (m, 2H), 1.47-1.67 (m, 11H), 1.19 (t, J=7.2 Hz, 3H);

MS (ESI): m/z 376 (29.5), 375 (M+H, 100).

Example 34 Synthesis ofN-Cycloheptyl-N′-(1-ethyl-pyrrolidin-2-ylmethyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine(130)

To 127 (5.009 g, 13.7 mmol) dissolved in THF (80 mL) was added asolution of 2-(aminomethyl)-1-ethylpyrrolidine (2.0 mL, 13.7 mmol) inTHF (10 mL) followed by addition of NaOH (5.5 mL, 2.5 N, 13.7 mmol) and13 mL of water. The reaction mixture was allowed to stir at reflux forabout 2 hours under N₂ atm. The reaction mixture was extracted 3 timeswith dichloromethane; the combined organic layers were washed with brineand dried over potassium carbonate. The sample was concentrated on therotary evaporator and the resulting solid was dried overnight undervacuum. Column chromatography (silica gel, 90:9:1dichloromethane:methanol:conc. ammonium hydroxide) yielded a lightyellow solid 130 (3.63 g, 58%), mp 76° C.; HPLC: Inertsil ODS-3V C18,40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 7.1 min,97.1% purity; MS (ESI): m/z 459 (16.5), 458 (M+H, 48.7), 362 (31.3), 250(100), 230 (22.8), 229 (62.7), 222 (17.2), 202 (34).

Example 35 Synthesis of2-[4-chloro-6-(3-chloro-4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-propane-1,3-diol(131)

To 101 (0.6114 g, 2 mmol) dissolved in acetone (3 mL) was added2-amino-propane-1,3-diol (0.1818 g, 2 mmol) dissolved in acetone (1 mL)and water (1 mL). Then water (1 mL) was added to the reaction mixturefollowed by 2.5 N NaOH (aq) (0.8 mL, 2 mmol). The reaction mixture washeated at reflux for 3 h under a N₂ atmosphere. The reaction mixture wasdiluted with ethyl acetate and washed 2×brine. The organic layer wasseparated, dried over anhydrous K₂CO₃, filtered, and concentrated underreduced pressure affording 0.634 g of a purple solid. The crude materialwas purified by silica gel flash column chromatography eluting with 100%ethyl acetate affording a colorless oil 131 (0.124 g, 18%); HPLC:Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264nm, R_(t) 5.7 min, 83.3% purity; MS (ESI): m/z 360 (M+H, 100), 338(10.7), 183 (10.3)

Example 36 Synthesis of2-{4-(3-chloro-4-methoxy-phenylamino)-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-propane-1,3-diol(132)

To 131 (0.979 g, 0.271 mmol) dissolved in 3 mL 1,4-dioxane was addedmethyl-4-(methylamino)piperidine (0.05 mL, 0.34 mmol) dissolved in 2 mL1,4-dioxane followed by the addition of 2.5 N NaOH (aq) (0.11 mL, 0.275mmol). The mixture was heated at reflux for 3 h 45 min, cooled to aboutroom temperature, and then concentrated under reduced pressure. Theresulting material was diluted with dichloromethane and filtered. Thefiltrate was then concentrated affording 56.5 mg of material. The crudematerial was purified by silica gel pipet column eluting with 100%methanol affording an white solid 132 (21.1 mg, 18%), mp 84° C.; MS(ESI): m/z 454 (34.7), 452 (M+H, 100), 422 (11.3), 248 (25.3), 247(51.3), 157 (60.3), 129 (27.5).

Example 37 Synthesis ofN-(1-benzyl-piperidin-4-yl)-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]-2,4,6-triamine(134)

To 133 (0.1252 g, 0.382 mmol, prepared as indicated herein) dissolved in3 mL acetonitrile was added N,N-diisopropyl ethyl amine (DIEA) (0.07 mL,0.382 mL) followed by 4-amino-1-benzylamine (0.07 mL, 0.382 mmol). Themixture was refluxed overnight under a N₂ atmosphere. The reactionmixture was diluted with methylene chloride and washed with brine. Theorganic layer was separated, dried over K₂CO₃, filtered and concentratedunder reduced pressure to afford 0.159 g of material. The crude materialwas purified by silica gel flash column chromatography eluting with96:3:1 methylene chloride:methanol:conc. ammonium hydroxide and thecollected fractions were dried over potassium carbonate, filtered andthen concentrated under reduced pressure to afford 77 mg of product. Asecond column under similar conditions was completed to afford anadditional 30 mg of material for a combined product 134 (103 mg, 50%);HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 13.7 min, 97.7% purity;

MS (ESI): m/z 538 (15.4), 536 (38.2), 448 (19.3), 446 (49.3), 290(41.4), 289 (84.6), 269 (100), 247 (4.4).

Example 38 Synthesis ofN²-(3-chloro-4-methoxy-phenyl)-N⁴-cycloheptyl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine(135)

To 134 (0.0485 g, 0.0867 mmol) in 2 mL methanol was added 10% Pd/C(0.052 g) followed by ammonium formate (0.0646 g, 1.02 mmol). Themixture was heated at reflux for about 1.5 h under a N₂ atmosphere. Thecooled reaction mixture was filtered by vacuum through Celite with amethylene chloride rinsing, and the filtrate concentrated under reducedpressure to afford 36 mg of material. The crude material was purified bysilica gel flash chromatography eluting with 90:9:1 methylenechloride:methanol:conc. ammonium hydroxide, and the collected fractionswere dried over potassium carbonate, filtered and then concentratedunder reduced pressure to afford a solid 135 (20 mg, 51.8%), mp 167° C.;HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 4.6 min, 52.1% (another major peak atR_(t) 7.3 min, 46.9%); MS (ESI): m/z 448 (4.4), 446 (12.5), 412 (22.7),386 (2.3), 265 (32.9), 248 (42.6), 244 (56.2), 228 (37.1), 227 (100),207 (6.9).

Example 39 Synthesis ofN²-(3-chloro-4-methoxy-phenyl)-N⁴-cycloheptyl-N⁶-(1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine(136)

To 133 (0.1257 g, 0.382 mmol, prepared as indicated herein) dissolved in3 mL acetonitrile was added DIEA (0.07 mL, 0.382 mL) followed by2-(aminomethyl)-1-ethyl pyrrolidine (0.06 mL, 0.382 mmol). The mixturewas refluxed overnight under a N₂ atmosphere. The reaction mixture wasdiluted with methylene chloride and washed with brine. The organic layerwas separated, dried over K₂CO₃, filtered and concentrated under reducedpressure to afford 0.143 g of material. The crude material was purifiedby silica gel flash column chromatography eluting with 96:3:1 methylenechloride:methanol:conc. ammonium hydroxide and the collected fractionsdried over approximately 1:1 potassium carbonate/sodium sulfate,filtered and then concentrated under reduced pressure to afford 77 mg ofproduct. A second column under similar conditions was completed toafford an additional 30 mg of material for a combined 98 mg (54%) of ayellow colored solid 136, mp 69-70° C.; HPLC: YMC Pack Pro C18, 40:30:30[KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 12.9 min, 96.5%purity; MS (ESI): m/z 476 (16.3), 474 (42.9), 260 (15), 259 (44.2), 258(100), 238 (56), 216 (5.3), 210 (9.2).

Example 40 Synthesis of2-chloro-4-(4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl-amino]-1,3,5-triazin-2-ylamino}-phenol(138)

Under anhydrous conditions, 137 (0.1008 g, 0.21 mmol, prepared asdescribed herein) in a dry round bottomed flask was dissolved inanhydrous methylene chloride (3 mL) under a N₂ atmosphere about 0° C.(ice/water bath) was added BBr₃ (2.1 mL, 2.1 mmol, 1 M in methylenechloride) slowly by syringe. The mixture was stirred for about 2 hoursat about 0° C. and then quenched with water (5 mL). After standingovernight at rt, the mixture was diluted with ethyl acetate, water and10% NaHCO₃ (aq), and the organic layer was separated then washed withbrine. The organic layer was then dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure to afford 0.648 g ofmaterial. The crude material was purified using silica gel flash columnchromatography eluting with 100% methanol to afford of a white solid 138(7 mg, 7%); HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 4.9 min, 90.3% purity; ¹H NMR (600 MHz,CDCl₃, 55° C.) (all resonances are broad) δ 7.93 (s, 1H), 7.13 (s, 1H),6.91-6.92 (m 1H), 6.55 (s, 1H), 4.80 (s, 1H), 4.59 (s, 1H), 4.02 (s,1H), 2.96-3.0 (m, 5H), 2.32 (s, 3H), 2.13 (s, 2H), 2.03 (s, 2H),1.86-1.88 (m, 2H), 1.53-1.67 (m, 12H); MS (ESI): m/z 463 (12.4), 461(27), 252 (59), 251 (100), 231 (32.3), 224 (1), 203 (9.8).

Example 41 Synthesis ofN²-cycloheptyl-N⁴-((S)-1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine(139)

To a mixture of cyanuric chloride (0.368 g, 2 mmol) in CH₃CN atapproximately −10 to −20° C. was added 3-fluoro-p-anisidine (0.28 g, 2mmol) in CH₃CN followed by the addition of N,N-diisopropylethylamine(DIEA) (0.35 mL, 2 mmol) and stirred for an hour. The reaction mixturewas then allowed to reach room temperature for an hour. The second stepwas continued without further purification. Cycloheptylamine (0.25 mL, 2mmol) and DIEA (0.35 mL, 2 mmol) were added and the reaction mixture wasstirred overnight at rt. The third step was also preceded without anyfurther purification. S-(−)-2-aminomethyl-N-ethyl pyrrolidine (0.29 mL,2 mmol) and DIEA (0.35 mL, 2 mmol) were added and the reaction mixturewas refluxed overnight. The reaction mixture was diluted with ethylacetate and washed with brine. The organic layer was separated and driedover potassium carbonate, filtered, and concentrated under reducedpressure affording 0.920 g crude material. The crude material waspurified by column chromatography to yield a white solid 139 (0.550 g,60%), mp 75-77° C.; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M,pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 7.9 min, 95.9% purity; MS (ESI): m/z458 (M+H, 100).

Example 42 Synthesis ofN²-cycloheptyl-N⁴-((R)-1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine(140)

To a mixture of cyanuric chloride (0.368 g, 2 mmol) in CH₃CN at about−10 to −20° C. was added 3-fluoro-p-anisidine (0.28 g, 2 mmol) in CH₃CNfollowed by the addition of N,N-diisopropylethylamine (0.35 mL, 2 mmol)and stirred for an hour. The reaction mixture was then allowed to reachroom temperature for an hour. Then cycloheptylamine (0.25 mL, 2 mmol)and DIEA (0.35 mL, 2 mmol) were added and the reaction mixture wasstirred overnight at rt. To this reaction mixtureR-(+)-2-aminomethyl-N-ethyl pyrrolidine (0.29 mL, 2 mmol) and DIEA (0.35mL, 2 mmol) were added and the reaction mixture was refluxed overnight.The reaction mixture was diluted with ethyl acetate and washed withbrine. The organic layer was separated and dried over potassiumcarbonate, filtered, and concentrated under reduced pressure affording0.920 g crude material. The crude material was purified by columnchromatography to yield a white solid 140 (0.500 g, 54.7%), mp 77-79°C.; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 7.9 min, 74.3% purity; MS (ESI): m/z458 (M+H, 100).

Example 43 Synthesis ofN²-cyclohexylmethyl-N⁴-((S)-1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine(141)

To a mixture of cyanuric chloride (0.368 g, 2 mmol) in CH₃CN at about−20° C. was added 3-fluoro-p-anisidine (0.28 g, 2 mmol) in CH₃CNfollowed by the addition of N,N-diisopropylethylamine (DIEA) (0.35 mL, 2mmol) and stirred for about 1 hour. The reaction mixture was thenstirred at room temperature for about 1 hour. Then, cyclohexylmethylamine (0.26 mL, 2 mmol) and DIEA (0.35 mL, 2 mmol) were added and thereaction mixture was stirred overnight at RT. Then,S-(−)-2-aminomethyl-N-ethyl pyrrolidine (0.29 mL, 2 mmol) and DIEA (0.35mL, 2 mmol) were added and the reaction mixture was refluxed overnight.The reaction mixture was diluted with ethyl acetate and washed withbrine. The organic layer was separated and dried over sodium sulfate,filtered, and concentrated under reduced. The crude material waspurified by column chromatography eluting with 96:3:1 methylenechloride:methanol:conc. ammonium hydroxide to yield a white solid 141(0.400 g, 43.7%), mp 68-69° C.; HPLC: Inertsil ODS-3V C18, 40:30:30[KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 8.2 min, 97.1%purity; MS (ESI): m/z 458 (M+H, 100), 362 (2.8), 230 (85.4).

Example 44 Synthesis ofN²-cyclohexylmethyl-N⁴-((R)-1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine(142)

To a mixture of cyanuric chloride (0.368 g, 2 mmol) in CH₃CN at about−20° C. was added 3-fluoro-p-anisidine (0.28 g, 2 mmol) in CH₃CNfollowed by the addition of DIEA (0.35 mL, 2 mmol) and stirred for about1 hour. The reaction mixture was then stirred at room temperature forabout 1 hour. Then, cyclohexylmethyl amine (0.26 mL, 2 mmol) and DIEA(0.35 mL, 2 mmol) were added and the reaction mixture was stirredovernight at room temperature. Then, R-(+)-2-aminomethyl-N-ethylpyrrolidine (0.29 mL, 2 mmol) and DIEA (0.35 mL, 2 mmol) were added andthe reaction mixture was refluxed overnight. The reaction mixture wasdiluted with ethyl acetate and washed with brine. The organic layer wasseparated and dried over sodium sulfate, filtered, and concentratedunder reduced pressure. The crude material was purified by columnchromatography eluting with 96:3:1 methylene chloride:methanol:conc.ammonium hydroxide to give 142 (0.100 g, 10.9%), mp 66-67° C.; HPLC:Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264nm, R_(t) 8.2 min, 96.7% purity; ¹H NMR (600 MHz, CDCl₃) δ 7.58-7.73(broad resonance, 1H), 7.07-7.11 (broad resonance, 1H), 6.82 (t, J=9 Hz,1H), 5.49-5.65 (broad resonance, 1H), 4.96-5.13(broad resonance, 1H),3.82 (s, 3H), 3.54-3.70 (broad resonance, 1H), 3.13-3.20 (br m, 4H),2.81 (broad resonance, 1H), 2.54 (broad resonance, 1H), 2.05-2.18 (m,2H), 2.01 (s, 1H), 1.50-1.83 (br m, 9H), 1.05-1.22 (m, 5H), 0.91 (apt q,J=11.4 Hz, 2H); MS (ESI): m/z 458 (M+H, 100), 362 (3.8), 230 (99.8), 216(1), 182 (1.1).

Example 45 Synthesis of({4-cycloheptylamino-6-[((S)-1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile(143)

To a mixture of cyanuric chloride (0.368 g, 2 mmol) in CH₃CN at about−20° C. was added N-phenyl glycinonitrile (0.264 g, 2 mmol) in CH₃CNfollowed by the addition of DIEA (0.35 mL, 2 mmol) and stirred for about1 hour. The reaction mixture was then stirred at room temperature forabout 1 hour. Then, cycloheptylamine (0.25 mL, 2 mmol) and DIEA (0.35mL, 2 mmol) were added and the reaction mixture was stirred overnight atrt. Then, S-(−)-2-aminomethyl-N-ethyl pyrrolidine (0.29 mL, 2 mmol) andDIEA (0.35 mL, 2 mmol) were added and the reaction mixture was refluxedovernight. The reaction mixture was diluted with ethyl acetate andwashed with brine. The organic layer was separated and dried over sodiumsulfate, filtered, and concentrated under reduced pressure. The crudematerial was purified by column chromatography eluting with 96:3:1methylene chloride:methanol:conc. ammonium hydroxide to yield 143,(0.300 g, 33%) mp 53-55° C.; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄(0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 6.9 min, 94.1% purity; MS(ESI): m/z 449 (M+H, 100), 381 (1.2), 353 (16.2), 226 (19.9), 225(54.3), 212 (20.5), 177 (18.3), 164 (9.6).

Example 46 Synthesis of({4-cycloheptylamino-6-[((R)-1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile(144)

To a mixture of cyanuric chloride (0.368 g, 2 mmol) in CH₃CN at about−20° C. was added N-phenyl glycinonitrile (0.264 g, 2 mmol) in CH₃CNfollowed by the addition of DIEA (0.35 mL, 2 mmol) and stirred for about1 hour. The reaction mixture was then stirred at room temperature forabout 1 hour. Then, cycloheptylamine (0.25 mL, 2 mmol) and DIEA (0.35mL, 2 mmol) were added and the reaction mixture was stirred overnight atrt. Then, R-(+)-2-aminomethyl-N-ethyl pyrrolidine (0.29 mL, 2 mmol) andDIEA (0.35 mL, 2 mmol) were added and the reaction mixture was refluxedovernight. The reaction mixture was diluted with ethyl acetate andwashed with brine. The organic layer was separated and dried over sodiumsulfate, filtered, and concentrated under reduced pressure. The crudematerial was purified by column chromatography eluting with 96:3:1methylene chloride:methanol:conc. ammonium hydroxide to yield 144,(0.300 g, 33%), mp 53-55° C.; HPLC: Inertsil ODS-3V C18, 40:30:30[KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 6.8 min, 92.6%purity; MS (ESI): m/z 449 (M+H, 100), 381 (1.4), 353 (11.8), 226 (13),225 (33.1), 212 (15), 177 (13.5), 164 (7.8).

Example 47 Synthesis ofN²-[(1-ethyl-2-pyrrolidinyl]-N⁴-(3-fluoro-4-methoxyphenyl)-6-[(S)-2-(methoxymethyl)-1-pyrrolidinyl]-1,3,5-triazine-2,4-diamine(145)

Cyanuric chloride (11.07 g, 60 mmol) was dissolved in 40 mL CH₃CN andwas cooled to about −20° C. To this was added DIEA (11.5 mL, 60 mmol)followed by 3-fluoro-4-methoxyaninline (8.47 g, 60 mmol) in 20 mL CH₃CN(reaction froze). The reaction was allowed to warm to room temperatureafter about 1 hour at −20° C. TLC (2% CH₃OH/CH₂Cl₂) and massspectroscopy indicated the presence of the compound 124. The reactionmixture was cooled to about 0° C. before adding DIEA (11.5 mL, 66 mmol).2-Aminomethyl-1-ethylpyrrolidine (7.77 g, 60 mmol) in CH₃CN (10 mL) wasadded. The reaction was allowed to warm to rt and stirred overnight.Then DIEA (11.5 mL, 66 mmol) and S-(+)-2-methoxyethylpyrrolidine (6.91g, 60 mmol) in 20 mL 1,4-dioxane were added. The reaction was heated atabout 50° C. overnight. The solvent was removed in vacuo, and theresulting residue was purified by flash chromatography on silica gelpacked in ethyl acetate. The front running impurities were removed andsubsequently the eluent was increased in polarity to 10% CH₃OH:ethylacetate. The material collected from the column was then dissolved inwater and extracted in CH₂Cl₂ (4 times), dried over MgSO₄, andconcentrated to dryness to give a brown solid 145 (9.7 g, 27.6% yield),71-72° C.; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 5.37 min, 90.3% purity; ¹H NMR (600MHz, CDCl₃, 55° C.) δ 7.69 (s, 1H), 7.08 (d, J=7.8 Hz, 1H), 6.86 (t, J=9Hz, 1H), 4.29 (s, 1H), 3.90-3.96 (m, 1H), 3.84 (s, 3H), 3.63-3.81 (m,6H), 3.35 (s, 3H), 3.23-3.25 (m, 1H), 2.85 (broad s, 1H), 2.78 (broad s1H), 2.14 (broad s, 2H), 1.89-2.04 (m, 6H), 1.37 (apparent t, J=7.2 Hz,3H); ¹³C NMR (150.8 MHz, CDCl₃, 55° C.) δ 165.8, 163.8 (2C), 152.3 (d,J_(c-f)=243.5 Hz), 143.0 (142.9, rotamer or diastereumer), 133.7(133.67, rotamer or diastereomer), 115.0, 114.4, 109.1 (108.9, rotameror diastereomer), 72.8, 66.6, 59.0, 57.0, 56.6, 53.7, 51.0, 46.8, 42.2,28.4 (28.2, rotamer or diastereomer), 23.1 (23.0, rotamer ordiastereomer), 10.9; MS (ESI) m/z 460.2 (M+H, 44.7), 251.1 (47.7), 235.1(27.5), 231.1 (37.4), 230.6 (100), 214.6 (36.5).

Example 48 Synthesis of(3-Chloro-4-methoxy-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine(101)

To cyanuric chloride (36.911 g, 200.0 mmol) dissolved in acetone (250mL) stirring at approximately 0-5° C. (ice-water bath), was added asolution of 3-chloro-p-anisidine (31.528 g, 200.0 mmol) in acetone (150mL) followed by the addition of NaOH solution (80 mL, 2.5 N, 200.0mmol). The reaction mixture was allowed to stir at approximately 0-5° C.(ice-water bath) for about 1 hour. The reaction mixture was then pouredover crushed ice and neutralized with 10% HCl (aq). The resulting solidwas washed with water and dried overnight under vacuum to afford 101(58.3 g, 96%), mp 165° C.; HPLC: YMC Pack Pro C18, 40:30:30 [KH₂PO₄(0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 24.3 min, 97.8% purity); MS(ESI): m/z 305 (M+H, 100), 283 (26.3), 271 (26.9), 269 (75.2), 139(16.2).

Example 49 Synthesis of6-Chloro-N-(3-chloro-4-methoxy-phenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine(133)

To a sample of compound 101 (20.02 g, 65.6 mmol) in acetone (200 mL) wasadded cycloheptylamine (8.3 mL, 65.5 mmol) in acetone (55 mL) slowly byaddition funnel at rt. Then water (66 mL) was added followed by aqueoussodium hydroxide (26.2 mL, 2.5 N, 65.5 mmol) by addition funnel. Thereaction mixture was heated at reflux under a nitrogen atmosphere forapproximately about 3 hours. The reaction was cooled, diluted with ethylacetate, washed 1 time with water, and finally 1 time with brine. Theorganic layer was separated and dried over potassium carbonate/sodiumsulfate. The organic layer was filtered and concentrated in vacuo. Theproduct (24.13 g) was purified by flash column chromatography (silicagel, 1:4 ethyl acetate:hexanes). The fractions were combined andconcentrated in vacuo to afford 133 as a pale yellow solid (17.66 g,70.5%), mp 146° C.; HPLC: Inertsil ODS-3V C18, 40:10:50 [KH₂PO₄ (0.01M,pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 58.8 min, 99.9% purity); MS (ESI):m/z 382 (M+H, 100), 241 (2.8), 226 (8.4), 139 (43.5), 116 (6).

Example 50 Synthesis ofN-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N′-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine (137)

To 133 (10.014 g, 26.2 mmol) in 1,4-dioxane (80 mL) was added slowlymethyl-(1-methyl-piperidin-4-yl)-amine (3.8 mL, 26.2 mmol) dissolved in1,4-dioxane (15 mL) by addition funnel. Then aqueous sodium hydroxide(10.5 mL, 2.5 N, 26.2 mmol) was added by addition funnel followed bywater (26 mL). The reaction mixture was heated at reflux for about 2.5hours under a nitrogen atmosphere. The reaction was cooled and dilutedwith methylene chloride. The reaction mixture was filtered using vacuumand the white solid 147 removed. The filtrate was then washed 1 timewith brine. The aqueous layer was back extracted 1 time with methylenechloride. The organic layers were combined and dried over potassiumcarbonate. The organic solution was filtered and concentrated in vacuoto afford the crude product (5.89 g). The crude reaction product waspurified by flash column chromatography (silica gel) eluting with 96:3:1methylene chloride:methanol:15 M ammonium hydroxide. The fractions werecombined, dried over sodium sulfate/potassium carbonate, filtered, andconcentrated in vacuo to afford 137 as a white solid (3.84 g, 30.9%), mp104-105° C.; HPLC: YMC Pack Pro C18, 40:30:30 [KH₂PO₄ (0.01M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 13.8 min, 97% purity); MS (ESI): m/z474 (M+H, 41), 408 (2.3), 364 (2.8), 258 (13), 239 (14), 239 (47.5), 238(100), 127 (5.3).

Example 51 Synthesis ofN²-(3-chloro-4-methoxy-phenyl)-N⁴-cycloheptyl-N⁶-methyl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine(146)

Compound 146 was isolated as a by-product via column chromatography(silica gel, 96:3:1 methylene chloride:methanol:conc. ammoniumhydroxide, mp 114-116° C.; TLC (silica gel, 90:9:1, CH₂Cl₂:CH₃OH; conc.NH₄OH), R_(f) 137 0.31 and R_(f) 146 0.15; HPLC: Inertsil ODS-3V C18,40:30:30 [KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 10.7 min,91.1% purity);

MS (ESI): m/z 460 (M+H, 25.4), 364 (17.9), 292 (2), 273 (17.1), 272(37.9), 252 (44), 251 (100), 231 (2.2), 157 (10.54), 118 (2.8).

Example 52 Synthesis of4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-1,3,5-triazin-2-ol(147)

Compound 147 was isolated as a by-product by vacuum filtration prior toisolation of 137, white solid, mp>310° C.; MS (ESI); m/z 727([2(363)+H], 1.2, 364 (M+H, 100).

Example 53 Synthesis ofN-(1-Aza-bicyclo[2.2.2]oct-3-yl)-N′-(3-chloro-4-methoxy-phenyl)-N″-)1-ethyl-pyrrolidin-2-ylmethyl)-[1,3,5]triazine-2,4,6-triamine(148)

To 101 (3.056 g, 10.0 mmol) dissolved in anhydrous acetonitrile (30 mL)at about 0° C. was added a solution of2-(aminomethyl)-1-ethylpyrrolidine (1.5 mL, 10.0 mmol) in anhydrousacetonitrile (5 mL) followed by addition of a DIEA (1.9 mL, 11.0 mmol).The reaction mixture was allowed to warm to room temperature and wasstirred at room temperature overnight under nitrogen. Then DIEA (1.9 mL,11 mmol) was added which was followed by addition of 3-aminoquinuclidinedihydrochloride (1.962 g, 10.0 mmol) in 1,4-dioxane (5 mL). The reactionmixture was allowed to stir at reflux overnight under nitrogen. Thereaction mixture was extracted 2 times with dichloromethane and 1 timewith ethyl acetate. The combined organic layers were washed one timewith brine and dried over anhydrous potassium carbonate. The organiclayer was with 20% HCl (aq). The aqueous layer was neutralized with 2.5N NaOH (aq) and then extracted 3 times with ethyl acetate. The combinedorganic layers were washed 1 time with brine, dried over potassiumcarbonate, concentrated on a rotary evaporator and allowed to dryovernight under vacuum. Column chromatography (silica gel, 85:14:1dichloromethane:methanol:conc. ammonium hydroxide) yielded a pale whitesolid 148 (100 mg, 2%), mp 83° C.; HPLC: Inertsil ODS-3V C18, 40:30:30[KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 8.1 min, 71.2%purity); MS (ESI): m/z 488 (M+H, 18.7), 280 (100), 245 ([M+2H]++, 37.4),236 (23.5).

Example 54 Synthesis ofN²-(3-chloro-4-diethylamino-phenyl)-N⁴-cycloheptyl-N⁶-(1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine(149)

To a mixture of cyanuric chloride (1.8 g, 9.7 mmol) in CH₃CN at about−20° C. was added 2-chloro-N,N-diethyl phenylene-1,4-diaminehydrochloride (2.35 g, 10 mmol) in CH₃CN followed by the addition ofN,N-diisopropylethylamine (DIEA) (1.75 mL, 10 mmol) and stirred for anhour. The reaction mixture was then allowed to reach room temperaturefor about 1 hour. Then cycloheptylamine (1.25 mL, 9.8 mmol) and DIEA(1.75 mL, 10 mmol) were added and the reaction mixture was stirredovernight at rt. Then, 2-(aminomethyl)-1-ethylpyrrolidine (1.45 mL, 10mmol) and DIEA (1.75 mL, 10 mmol) were added and the reaction mixturewas refluxed overnight. The reaction mixture was diluted with ethylacetate and washed with brine. The organic layer was separated and driedover sodium sulfate, filtered, and concentrated under reduced pressure.The crude material was purified by column chromatography (silica gel)eluting with 96:3:1 methylene chloride:methanol:conc. ammonium hydroxideto yield 149 (0.800 g, 15%) as a white solid, mp 84-85° C.; HPLC:Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264nm, R_(t) 9.5 min, 96% purity; MS (ESI): m/z 515 (M+H, 9.4), 259 (16.8),258 (55.1), 257 (100).

Example 55 Synthesis ofN²-cycloheptyl-N⁴-(2-dimethylamino-ethyl)-N⁶-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine(150)

Cyanuric chloride (1.84 g, 10 mmol) in CH₃CN (20 mL) was cooled to about−10° C. was added 3-fluoro-p-anisidine (1.41 g, 10 mmol) followed byDIEA (1.8 mL, 10 mmol). The reaction was stirred for about 45 min thenat room temperature for about 45 min under an N₂ atmosphere.Cycloheptylamine (1.26 mL, 10 mmol) was added followed by DIEA (1.8 mL,10 mmol) and the reaction was stirred at room temperature overnight.N,N-dimethylethylenediamine (1.1 mL, 10 mmol) was added followed by DIEA(1.8 mL, 10 mmol) and the mixture was heated at reflux under N₂overnight. The reaction was diluted with ethyl acetate, washed withbrine, and dried over anhydrous K₂CO₃. The material (1.178 g) waspurified by silica gel column chromatography to afford a solid 150(1.178 g, 28%), mp 73-76° C.; HPLC: Inertsil ODS-3V C18, 40:30:30[KH₂PO₄ (0.01M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 10.8 min, 95.1%purity; MS (ESI): m/z 418 (M+H, 100), 373 (11.9), 322 (7.8), 277 (6.8),162 (3.6).

Example 56 Synthesis of({4-cycloheptylamino-6-[1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile(151)

To cyanuric chloride (1.84 g, 10 mmol) in CH₃CN (20 mL) at about −10 to−20° C. was added DIEA (1.75 mL, 10 mmol) and N-phenyl glycinonitrile(1.3 g, 10 mmol), and stirred for about 1 hour. The reaction mixture wasthen allowed to reach room temperature for an hour. To this reactionmixture, DIEA (1.75 mL, 10 mmol) and cycloheptylamine (1.25 mL, 10 mmol)were added and the reaction mixture was stirred overnight at rt. Then,DIEA (1.75 mL, 10 mmol) and 2-aminomethyl-N-ethylpyrrolidine (1.45 mL,10 mmol) were added and the reaction mixture was refluxed overnight. Thereaction mixture was worked-up, isolated, and then purified by columnchromatography (silica gel) eluting with 96:3:1 methylenechloride:methanol:conc. ammonium hydroxide to yield 151, (3 g, 66%), mp52-54° C.; MS (ESI): m/z 449 (M+H, 100), 225 [(M+2H)²⁺, 22.3].

Example 57 Synthesis ofN-Azepan-1-yl-6-chloro-N′-(3-chloro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine(152)

To 101 (6.03 g, 20.0 mmol) dissolved in acetone (75 mL) was added asolution of 1-aminohomopiperidine (2.3 mL, 20.0 mmol) in acetone (10 mL)followed by addition of NaOH (8.0 mL 2.5 N NaOH solution, 20.0 mmol) and20 mL of water. The reaction mixture was allowed to stir at refluxovernight under nitrogen. The reaction mixture was extracted 3 timeswith dichloromethane; the combined organic layers were washed with brineand dried over potassium carbonate. The sample was concentrated on arotary evaporator and the resulting oil was dried overnight undervacuum. Column chromatography (96:3:1 dichloromethane:methanol:conc.ammonium hydroxide) yielded a light purple solid 152 (1.2 g, 16%), mp139° C.; TLC (silica gel, 96:3:1, CH₂Cl₂, CH₃OH, conc. NH₄OH), R_(f)0.31; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 52.5 min, 94.9% purity; MS (ESI): m/z383 (M+H, 100).

Example 58 Synthesis ofN″-(3-chloro-4-methoxy-phenyl)-N,N′-bis-perhydro-azepin-1-yl-1,3,5-triazine-2,4,6-triamine(153)

Compound 153 was isolated as a by-product (2.3 g) by columnchromatography (silica gel, 96:3:1, CH₂Cl₂, CH₃OH, conc. NH₄OH), mp 199°C.; TLC (silica gel, 96:3:1, CH₂Cl₂, CH₃OH, conc. NH₄OH), R_(f) 0.11;HPLC: Inertsil ODS 3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 15 min, 86% purity); MS (ESI): m/z 461(M+H, 100), 366 (19.7), 365 (19.6), 232 (11), 231 (27.3).

Example 59 Synthesis ofN-Azepan-1-yl-N′-(3-chloro-4-methoxy-phenyl)-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine(154)

To 152 (0.2007 g, 0.5 mmol) dissolved in THF (10 mL) was added asolution of N-methyl-4(methylamino)piperidine (0.07 mL, 0.5 mmol) in THF(1 mL) followed by the addition of DIEA (1.0 mL, 0.55 mmol) inacetonitrile (1 mL). The reaction mixture was allowed to stir at refluxovernight under nitrogen. The reaction mixture was extracted 3 timeswith dichloromethane; the combined organic layers were washed with brineand dried over potassium carbonate. The sample was concentrated on arotary evaporator and the resulting oil was dried overnight undervacuum. Column chromatography (90:9:1 dichloromethane:methanol:conc.ammonium hydroxide) yielded a light yellow solid 154 (65 mg, 27%), mp100° C.; TLC (silica gel, 90:9:1 CH₂Cl₂:CH₃OH, conc. NH₄OH), R_(f) 0.36;MS (ESI): m/z 475 (M+H, 23.2), 378 (11.6), 258 (68.9), 239 (52.2), 238(100).

Example 60 Synthesis ofN⁴-(3-chloro-4-methoxy-phenyl)-N⁶-methyl-N²-perhydro-azepin-1-yl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine(155)

Compound 155 was obtained as a by product (50 mg) of the reaction viacolumn chromatography (silica gel, 90:9:1 dichloromethane:methanol:conc.ammonium hydroxide), mp 81° C.; TLC (silica gel, 90:9:1 CH₂Cl₂:CH₃OH,conc. NH₄OH), R_(f) 0.25; MS (ESI): m/z 461 (M+H, 20.3), 430 (2.8), 273(11.8), 272 (25.5), 251 (100), 236 (4.6), 215 (4.7).

Example 61 Synthesis ofN,N′-di-n-propyl-N″-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine(156)

To cyanuric chloride (0.368 g, 2 mmol) in CH₃CN at about −20° C. wasadded 3-fluoro-p-anisidine (0.28 g, 2 mmol) in CH₃CN followed by theaddition of DIEA (0.39 mL, 2.2 mmol) and stirred for about 1 hour. Thereaction mixture was then stirred at room temperature for about 1 hour.Then n-propylamine (1.64 mL, 19.9 mmol) and DIEA (0.39 mL, 2.2 mmol)were added and the reaction mixture was stirred overnight at rt. Thereaction mixture was worked up as usual, diluted with ethyl acetate andwashed with brine. The organic layer was separated and dried over sodiumsulfate, filtered, concentrated under reduced pressure, and compound 156was purified by silica gel column chromatography.

mp 53-55° C.; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 12.6 min, 93.7% purity; MS (ESI): m/z335 (M+H, 100), 331 (1.5), 126 (1).

Example 62 Synthesis ofN,N′-dicyclopropyl-N″-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine(157)

To cyanuric chloride (0.368 g, 2 mmol) in CH₃CN at about −20° C. wasadded 3-fluoro-p-anisidine (0.28 g, 2 mmol) in CH₃CN followed by theaddition of DIEA (0.39 mL, 2.2 mmol) and stirred for about 1 hour. Thereaction mixture was then stirred at room temperature for about 1 hour.Then cyclopropylamine (1.39 mL, 20 mmol) and DIEA (0.39 mL, 2.2 mmol)were added and the reaction mixture was stirred overnight at rt. Thereaction mixture was worked up as usual, diluted with ethyl acetate andwashed with brine. The organic layer was separated and dried over sodiumsulfate, filtered, concentrated under reduced pressure, and compound 157was purified by silica gel column chromatography (200 mg, 30%), mp91-92° C.; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 8.6 min, 99.1% purity; MS (ESI): m/z331(M+H, 100), 305 (0.8), 151 (0.3).

Example 63 Synthesis ofN²-Cycloheptyl-N⁴-(3-fluoro-4-methoxy-phenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine(158)

To cyanuric chloride (0.180 g, 1 mmol) in 1,4-dioxane (1 mL) at about−10 to −20° C. was added N,N-diisopropylethylamine (DIEA) (0.19 mL, 1mmol) in CH₃CN (1 mL) and 3-fluoro-p-anisidine (0.14 g, 1 mmol) in CH₃CN(1 mL) and stirred for about 1 hour. The reaction mixture was thenstirred at room temperature for about 1 hour. Then a solution ofcycloheptylamine (0.13 mL, 1 mmol) and DIEA (0.19 mL, 1 mmol) in CH₃CN(0.5 mL) was added and the reaction mixture was stirred overnight at rt.Then, N-methyl-4(methylamino)piperidine (0.15 mL, 1 mmol) and DIEA (0.19mL, 1 mmol) in CH₃CN (0.5 mL) were added and the reaction mixture wasrefluxed overnight. The reaction mixture was worked-up using saturatedsodium bicarbonate, and brine. The organic layer was separated and driedover sodium sulfate, filtered, and concentrated under reduced pressure.The crude material was purified by column chromatography (silica gel,90:9:1 dichloromethane:methanol:conc. ammonium hydroxide) to give 158(0.130 g, 28%); TLC (silica gel, 90:9:1, CH₂Cl₂, CH₃OH, conc. NH₄OH),R_(f) 0.26); ¹H NMR (600 MHz, CDCl₃, 55° C.) δ 7.74 (br s, 1H), 6.94 (brs, 1H), 6.81-6.84 (m, 2H), 4.83 (br resonance, 1H), 4.55 (s, 1H), 3.98(s, 1H), 3.82 (s, 3H), 2.97 (s, 3H), 2.94 (br d, J=11.9 Hz, 2H), 2.29(s, 3H), 2.06-2.10 (m, 2H), 1.93-1.97 (m, 2H), 1.84-1.90 (m, 2H),1.44-1.66 (m, 12H).

Example 64 Synthesis ofN²-Cycloheptyl-N⁴-(3-fluoro-4-methoxy-phenyl)-N⁶-methyl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine(159)

Compound 159 was isolated as a by-product (55 mg) by columnchromatography (silica gel, 90:9:1 dichloromethane:methanol:conc.ammonium hydroxide); TLC (silica gel, 90:9:1, CH₂Cl₂, CH₃OH, conc.NH₄OH), R_(f) 0.1); HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M,pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 8.3 min, 93.5% purity; MS (ESI): m/z443 (M+H, 100).

Example 65 Synthesis ofN²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine,hydrogen chloride salt (160)

To 171 in dry methanol (1 mL, prepared according to parallel synthesisMethod C using the appropriate monomers, as disclosed herein) was addedHCl (0.3 mL, 0.3 mmol, 1 M in diethyl ether) by syringe under a N₂atmosphere. The mixture was stirred for 10 min at room temperature,concentrated and dried in vacuo overnight to give an off-white solid 160(0.131 g) that is water soluble, mp 189-190° C. (at 160° C. sample turnsbrown); HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 7.3 min, 89.1% purity.

Example 66 Synthesis of[N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]trizaine-2,4,6-triamine(161)

To 137 (0.473 g, 1.0 mmol) dissolved in methanol (5 mL) was added 1.0 Mhydrochloric acid in diethyl ether (1.0 mL, 1 mmol). The reactionmixture was allowed to stir for about 1 hour at room temperature. Thereaction mixture was then concentrated on a rotary evaporator. Theresulting solid was dissolved in water, filtered and concentrated on therotary evaporator. The sample was freeze dried under vacuum and a solid161 (359.1 mg, 70%) was collected, mp 173-176° C.

Example 67 Synthesis ofN²-(3-chloro-4-diethylamino-phenyl)-N⁴-cycloheptyl-N⁶-(1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triaminehydrogen chloride salt (163)

To 162 (1.0 g, 2 mmol, prepared according to parallel synthesis method Awith the appropriate monomers, as disclosed herein) in methanol (10 mL)was added HCl (2.5 mL, 2.5 mmol, 1 M) in diethyl ether and stirred. Thereaction mixture was evaporated. It was then dissolved in water,filtered, evaporated in vacuo, and dried over night under vacuum toafford a solid 163 (1.1 g, 93%).

Example 68 Synthesis ofN²-cycloheptyl-N⁴-(1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triaminehydrogen chloride salt (164)

To 130 (2.285 g, 5 mmol) in dry methanol (10 mL) was added HCl (5 mL, 5mmol, 1 M in diethyl ether) and stirred at room temperature for about 1hour. The reaction was evaporated in vacuo, dissolved in water,filtered, evaporated and then dried under vacuum overnight to afford asolid 164 (2.396 g, 97%), mp 131-133° C.; HPLC: Inertsil ODS-3V C18,40:30:30 [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 7.9 min,98.2% purity.

Example 69 Synthesis ofN²-(cyclohexylmethyl)-N⁴-[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶-(4-fluoro-3-methoxyphenyl)-1,3,5-triazine-2,4,6-triaminehydrogen chloride salt (165)

To 136 (0.457 g, 1 mmol) in dry diethyl ether was added HCl (1 mL, 1mmol, 1 M in diethyl ether). A precipitate formed immediately. Themixture was stirred at room temperature for about 1 hour, and thenconcentrated in vacuo. The resulting material was dissolved in water,filtered, evaporated, and dried overnight in vacuo to give a solid 165(0.400 g, 81%), mp 85° C.; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄(0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 8.2 min, 89.6% purity;

Example 70 Synthesis of({4-cycloheptylamino-6-[(1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrilehydrogen chloride salt (166)

To 151 (0.448 g, 1 mmol) in dry diethyl ether (2 mL) was added HCl (1mL, 1 mmol, 1 M in diethyl ether). The mixture was stirred at roomtemperature for about 1 hour, and then concentrated in vacuo. Theresulting material was dissolved in water (5-10 mL), filtered,evaporated, and dried overnight under vacuum to give a solid 166 (0.418g, 86%), mp 125-127° C.; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄(0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 6.9 min, 73.4% purity.

Example 71 Synthesis ofN²-cycloheptyl-N⁴-(3-fluoro-4-methoxy-phenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triaminemaleate salt (167)

Compounds 158 (100.3 mg, 0.219 mmol) and maleic acid (25.4 mg, 0.219mmol) were dissolved in CH₃OH (2 mL) and stirred at room temperatureunder a N₂ atmosphere for about 75 ml. The reaction mixture was filteredthrough a cotton plug and concentrated in vacuo to afford a solid 167,0.1239 g, mp 99-100° C. In a qualitative test, this material waswater-soluble. HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 7.7 min 87.9% purity.

Example 72 Synthesis ofN²-cycloheptyl-N⁴-(3-fluoro-4-methoxy-phenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triaminecitrate salt (168)

Compounds 158 (100 mg, 0.219 mmol) and citric acid (42.1 mg, 0.219 mmol)were dissolved in CH₃OH (2 mL) and stirred at room temperature under aN₂ atmosphere for about 2 hours. The reaction mixture was filteredthrough a cotton plug and concentrated in vacuo to afford a solid 168(0.1387 g), mp 125° C. In a qualitative test, this material was waterinsoluble. HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 7.7 min, 90.1% purity.

Example 73 Synthesis ofN²-cycloheptyl-N⁴-(3-fluoro-4-methoxy-phenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triaminesuccinate salt (169)

Compounds 158 (101.5 mg, 0.219 mmol) and succinic acid (24.8 mg, 0.219mmol) were dissolved in CH₃OH (2 mL) and stirred at room temperatureunder a N₂ atmosphere for about 75 min. The reaction mixture wasfiltered through a cotton plug and concentrated in vacuo to afford asolid 169 (0.1248 g), mp 81° C. In a qualitative test, this material waswater-soluble. HPLC: Inertsil ODS-3V C18, 40:30:30 [KH₂PO₄ (0.01M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 7.6 min, 89.8% purity.

Example 74 Synthesis ofN-(3-Bromo-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triaminehydrogen chloride salt (170)

To 123 (1.0 mmol) dissolved in methanol (5 mL) was added 1.0 Mhydrochloric acid in diethyl ether (1.0 mL, 1 mmol). The reactionmixture was allowed to stir for about 1 hour at room temperature. Thereaction mixture was then concentrated on a rotary evaporator. Theresulting solid was dissolved in water, filtered and concentrated on therotary evaporator. The sample was freeze dried under vacuum and a solid170 (70%) was collected

Example 75 Alternative Synthetic Route to Tris(amino) 1,3,5-TriazineCompounds

The following reaction scheme represents a proposed and alternativesynthetic routes to 1,3,5-triazines.

This scheme represents a modification of the synthetic route describedin the patent to prepare the tris-amino substituted 1,3,5-triazines.Alternative leaving groups, X, could be used as compared to cyanuricchloride (X=Cl) in the S_(N)Ar reaction with a sequential addition of anucleophilic amine in the presence of an acid (proton) scavenger toafford the tris-substituted 1,3,5-triazine with the desired combinationof amino groups.

Example 76 Alternative Synthetic Route to Tris(amino) 1,3,5-TriazineCompounds

The following reaction scheme represents a proposed and alternativesynthetic routes to 1,3,5-triazines.

This scheme represents a modification of the synthetic route describedin the patent text to prepare the tris-amino substituted1,3,5-triazines. Bases, including excess amine reagent R₂NH, could beused as acid (proton) scavengers alternatively to the Hünig's base(iPr₂NEt) used routinely in our procedure. These bases can include otherorganic tertiary amine bases or ionic, inorganic bases. One can usestrong bases (NaH, KH, or RLi) to first deprotonate the amino monomerbefore addition to the cyanuric-X substrate. Additionally, one can use asolid supported base (e.g., resin-NR₂, a modified Hünig's base) as aproton scavenger. This potentially enables an easier isolation procedureand cleaner reaction products. Logically, one would use the appropriatesolvent or combination of solvents that is compatible with the base ofchoice for this procedure.

Example 77 Alternative Synthetic Route to Tris(amino) 1,3,5-TriazineCompounds

The following reaction scheme represents a proposed and alternativesynthetic routes to 1,3,5-triazines.

This scheme represents a modification of the synthetic route describedin the patent to prepare the tris-amino substituted 1,3,5-triazines.Using melamine as the starting material, the method outlined wouldinvolve three sequential reductive amination procedures. With control ofaddition, temperature, and pH, the choice of aldehydes or ketone, onecan prepare tris-amino substituted triazines with the desiredcombination of amino groups.

Example 78 Alternative Synthetic Route to Tris(amino) 1,3,5-TriazineCompounds

The following reaction scheme represents a proposed and alternativesynthetic routes to 1,3,5-triazines.

This scheme represents a solid phase synthetic approach to preparingsymmetrically or asymmetrically substituted tris-amino substituted1,3,5-triazines. The resin should possess a readily cleavable linkergroup (L) and a leaving group (G) for attachment of an amino group. Thescheme outlines the synthesis by initially attaching a simple aminogroup, NH₂, by reacting the resin with ammonia. Using standard, S_(N)Archemistry for substitution of a perhalogenated 1,3,5-triazine, thetriazine can be attached to the aminated resin. Sequential substitutionsof the halogens on the triazine core with functionalized amines in thepresence of an acid scavenger will produce the desired di-aminosubstituted 1,3,5-triazine. Cleavage of the triazine from the resintether will afford the tris-amino substituted triazine product. The freeNH₂ moiety of the triazine can be further alkylated or functionalizedusing standard chemistry such reductive amination or N-alkylation togive a completely functionalized tris-amino substituted 1,3,5-triazine.

Example 79 Alternative Synthetic Route to Tris(amino) 1,3,5-TriazineCompounds

The following reaction schemes (Schemes A and B) represent proposed andalternative synthetic routes to 1,3,5-triazines.

These schemes represent variations on using the Suzuki coupling tosynthesize tris-amino substituted 1,3,5-triazines. As illustrated inScheme A, one can sequentially react the amino groups of melamine withan alkyl or aryl boronic acid derivative in the presence of theappropriate palladium catalyst, additives and solvent to afford thesymmetric or asymmetric tris-amino substituted 1,3,5-triazines similarto previously described examples. In Scheme B, a tris-boronic acid1,3,5-triazine can be prepared from cyanuric chloride or bromide. Thisderivative can then be coupled with an aryl or alkyl amine, asillustrated in previous amine monomer descriptions, in the presence ofthe appropriate metal catalysts (e.g., Cu or Pd catalyst), additives andsolvent to afford the symmetric or asymmetric tris-amino substituted1,3,5-triazines.

Example 80 Proteoglycan Induction

Smooth muscle cells reach quiescence during serum starvation resultingin a blockade of DNA synthesis. To demonstrate the role of perlecan(proteoglycan example) in SMC quiescence, cells were starved by removingserum from the media. The cells used in this Example and the otherexamples herein were human aortic SMC, grown in basal mediumsupplemented with growth factors, bFGF and epidermal growth factor (EGF)(Clonetics, San Diego, Calif.).

SMC secretion of total PGs (proteoglycans) as well as perlecan weredetermined in the presence or absence of one or more compounds of thepresent invention. PGs were radiolabeled with (³⁵S)sulfate by incubatingthe cells with (³⁵S)sulfate for 2 to 6 hours. Media PGs were collectedand purified by DEAE-cellulose chromatography. Cell-associated PGs wereassessed by extracting cells with 50 mM Tris buffer pH 7.4 containing 4M urea, 1% Triton X-100, 0.1 mM EDTA and 1 mM PMSF. Aqueous solutions of(³⁵S)sulfate and (³H)leucine were from Amersham. Control cells have noadded compounds whereas treated cells have one or more compounds of thepresent invention added.

To determine changes in PG levels, DEAE-cellulose chromatography wasperformed. A DEAE-cellulose column was equilibrated with 50 mM Trisbuffer pH 7.4 containing 4 M urea, 0.1 M NaCl, 0.1 mM EDTA, 1 mM PMSFand 1% 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).The column was washed with the same buffer and buffer containing 0.25 MNaCl and PG were eluted with the same buffer containing 0.5 M NaCl.Fractions containing radioactivity (³⁵SO₄) were pooled and dialyzedagainst MEM overnight and counted.

To determine the relative proportion of HSPG and chondroitinsulfate/dermatan sulfate proteoglycan (CS/DS PG), an aliquot of thepooled fraction was incubated in 50 mM sodium acetate buffer pH 5.2 with1 unit/ml each of heparanase and heparitinase or with 0.5 units ofchondroitin ABC lyase for 16 h at 37° C. Chondroitan ABC refers todifferent isomeric types of chondroitin, e.g. chondroitin A, chondroitinB, and chondroitin C. The reaction mixture was precipitated either with0.5 volumes of 1% cetyl pyridinium chloride or with 3 volumes of ethanolto precipitate undigested glycosaminoglycans. Radioactivity in thesupernatant and pellet was determined.

To determine changes in perlecan protein in response to the presence ofa compound, cells were grown in serum-free or serum-containing media inthe presence of (³H)leucine for 24 h (steady state). Cells were platedat low density (8×10⁴/well in 48 well plate, 30-40% confluency) andcultured for 24 h (hour). Wells were then replenished with fresh mediumcontaining no serum or 10% fetal bovine serum (FBS). Following another24 h incubation, cells were labeled with (³H)thymidine for 6 h andradioactivity incorporated into the DNA was determined bytrichloroacetic acid (TCA) precipitation of the cell lysate.(³H)thymidine was from NEN. Purified PG (0.5 M eluate) wereimmunoprecipitated by incubation with an anti-perlecan antibody(100-fold diluted) followed by precipitation with Protein A-Sepharose.Immunoprecipitates were analyzed by 5% SDS-PAGE. Perlecan (Mr>550 kDa)was identified by autoradiography. Control cells have no addedcompound(s) whereas treated cells have one or more compounds of thepresent invention added.

Example 81 Inhibition of Smooth Muscle Cell Proliferation

Purified perlecan from SMC medium by DEAE-cellulose chromatography wasobtained using methods in Example I, and was tested for itsantiproliferative effects on SMC.

The addition of perlecan to serum-containing medium inhibited SMC growthby 70%. Sub-confluent SMC (40-50% confluence) were incubated inserum-free medium or 10% serum-containing medium with or withoutpurified perlecan for 24 h. DNA synthesis was then determined byincubating cells for another 5 h in medium containing (³H)thymidine. TCAprecipitable (DNA) thymidine counts were determined and expressed aspercentage of DNA synthesis in cells grown in 10% FBS.

This assay can be used to show the effect of a compound on perlecandirectly by incubating the compound to perlecan first, then performingthe assay. Alternatively, the cells can be pretreated with at least onecompound of the present invention to show indirect effects. Controlcells have no added compounds whereas treated cells have one or morecompounds of the present invention added.

Example 82 Triazine Compounds in Smooth Muscle Cell Proliferation Assay

Human aortic smooth muscle cells (Clonetics) were used. Cells were grownin basal medium containing 5% fetal bovine serum supplemented withgrowth factors, basic fibroblast growth factor, epidermal growth factorand insulin. To determine the effects triazine compounds of the presentinvention had on SMC proliferation, cells were plated at low density(4000 cell per well in a 96 well plate) and cultured for 24 h. The cellswere then serum starved for 24 h to induce quiescence. Fresh growthmedium containing no compound or 10 μM compound was then added andfurther incubated for 24 h. Cell number was determined by using a cellproliferation assay kit (Celltiter96 AQ_(ueous) from Promega).

The effects of different triazine compounds on smooth muscle cellproliferation are shown in FIG. 53. Many of the triazine compoundsinhibited SMC proliferation by greater than 70%.

Example 83 Induction and Measurement of Endothelial Heparanase Protein

Experiments were carried out on human microvascular endothelial cells(HMVEC) grown in 48-well plates (−90% confluency). To induce heparanaseactivity, culture media was replaced with 200 μl Dulbecco's ModifiedEagle's medium (DMEM) complemented with 1% bovine serum albumin (BSA)and with or without stimulants (5 ng/ml TGF-alpha, 1 ng/ml IL 1alpha,200 ng/ml VEGF or other stimulants, cytokines, or inducers as required).The secreted proteins were analyzed by SDS/PAGE and heparanase proteinwas detected by immunoblotting using polyclonal anti-human heparanaseantibody. The changes of heparanase expression determined bydensitometric analysis. The induction and measurement of endothelialheparanase protein reported in the Tables herein were carried outaccording to this Example.

Example 84 Preparation of Biotinylated HS

Heparan sulfate (HS) was biotinylated using biotin with extended spacerarms using succinimidyl-6-(biotinamido) hexanoate (NHS-LC-Biotin)obtained from Pierce. About 0.5 ml HS solution (2 mg/ml in NaHCO3, pH8.5) was mixed with 0.05 ml of a freshly prepared solution ofNHS-LC-Biotin in dimethyl sulfoxide. The mixture was incubated at roomtemperature for 1 hour. Unconjugated biotin was removed bycentrifugation (10,000 RPM) through Microcon-3 filter (Millipore)followed by dilution with phosphate buffered saline (PBS). Thisprocedure was repeated five times to ensure complete removal of freebiotin. Unwanted aldehydes in the reaction were then quenched byincubation with one milliliter of Tris-glycine buffer (25 mM-183 mM, pH8.3) at room temperature for 20 minutes. The mixture was subjected tothree rounds of microfiltration as described above. Biotinylated HS (5mg/ml in PBS) was aliquoted and stored at −20° C. To obtain maximumbiotinylation, a 25-fold molar excess of biotin was used. Using HABAreagent, it was determined that the ratio of HS to biotin was 1:2.

The extent of biotinylation of HS was determined using Avidin-HABA(Pierce Chemical Co). The HABA assay can be used over a wide range of pHand salt concentrations. HABA (4-hydroxyazobenzene-2′-carboxylic acid)is a dye that binds to avidin and can serve as an indicator ofunoccupied binding sites. Avidin combines stoichiometrically withbiotin, making it possible to use any physiochemical differences betweenavidin and the avidin-biotin complex as the basis of a qualitative andquantitative assay method for either component.

When HABA binds to avidin, there is a large spectral change in the HABAdye. A new absorption band appears at 500 nm, which is characteristic ofthe quinoid form of the dye. The avidin-biotin complex does not bindHABA and because the dissociation constant of the complex is so low, thedye is stoichiometrically displaced by biotin. Consequently, the HABAassay can be the basis of both colorimetric and titrimetric assays. Theamount of avidin can be calculated directly from the increasedabsorbance at 500 nm, or the dye may be used as an indicator in aspectrophotometric titration with biotin.

The absorption band that results from the avidin-HABA complex decreasesproportionately when biotin is added. Since biotin has such a highaffinity for avidin, it displaces the HABA dye. The unknown amount ofbiotin can be determined by preparing a standard curve using knownamounts of biotin to displace the HABA which bound to avidin, andplotting against the absorbance at 500 mu.

HABA solution was prepared by adding 24.2 mg of HABA (Pierce) to 9.9 mlH20, and then adding 0.1 ml 1 M NaOH. Avidin-HABA reagent was preparedby adding 10 mg of avidin and 600 gl of HABA solution to 19.4 ml ofphosphate buffered saline. To 1 ml of Avidin-HABA reagent in a cuvette,100 μl of biotinylated HS was added, and the optical density wasmeasured at 500 nm in a spectrophotometer. A standard curve wasdetermined using known amounts of HABA. The decrease in optical densityof the HABA following the addition of biotinylated HS was determined.

Example 85 Heparanase Assay

Biotin-labeled HS made as described above was digested with heparanase,under both control and treated conditions, and the reaction containingundegraded and degraded HS was bound to in a biotin-binding plate.Streptavidin, conjugated with an enzyme, was added to the binding plate.Quantitation of the color reaction measured the amount of availablebiotin binding sites. A decrease in color from a known amount reflectsHS digestion by heparanase. Control conditions have no added compound ofthe present invention, and treated conditions have compounds of thepresent invention added.

A lyophilized powder of heparanase (heparanase III obtained fromSeikagaku) containing 0.1 units of enzymatic activity was hydrated in100 μl of Reaction Buffer (3.33 mM calcium acetate pH 7.0, containing0.1 mg/ml BSA). This solution was then diluted to a workingconcentration of heparanase solution (0.01 micro-units to 1 milli-unit)in Reaction Buffer. Enzyme activity was defined by the manufacturer ofthe heparanase (Seikagaku) as follows: one unit of enzyme activity isdefined as amount required to generate 1 micromole of hexuronic acid perminute. Biotin-HS was diluted to a desired concentration in ReactionBuffer.

To determine heparanase activity, 10 μl of heparanase solution, with orwithout at least one of the compounds of the present invention, wasmixed with 200 μl of the biotin-HS substrate in a 96 well plate. Thereaction was incubated at 43° C. for 1 hour. One hundred microliters ofthe reaction mixture was added to a hydrated biotin-binding plate(Chemicon) and incubated at 37° C. for 30 minutes. The biotin-bindingplates were hydrated with 200 μl of 1× Assay Buffer (Chemicon). Wellswere washed five times with 1× Assay Buffer and incubated with 100 μl of1:3000 diluted Streptavidin-Enzyme Conjugate (Chemicon) for 30 minutesat 37° C. The wells were washed five times with 1× Assay Buffer andincubated for 20 minutes with 100 μl of Substrate Solution (Chemicon).Color development in the wells was assessed by measuring the opticaldensity at 450 nm in a microplate reader (Labsystems, Muliskan Ascentmodel). Differences between the control and the treated conditionsindicate the heparanase modulating activity of the added compound orcompounds.

Example 86 AGE-Induced Inflammatory Response Determined by IL-6 ELISA

Human aortic endothelial cells (HAEC, Clonetics) were cultured accordingto manufacturer in growth medium (Clonetics): basal medium containinghuman epidermal growth factor, hydrocortisone, vascular endothelialgrowth factor, heparin binding growth factor-B, long R3-insulin-likegrowth factor-1, ascorbic acid, gentamicin/amphotericin and 5% FBS.These cell were allowed to reach at least 90% confluency beforesubjected to experimental treatments. Glycated human serum albumin(G-HSA) was from US Biologicals. Tumor necrosis factor α was from R&DSystems.

Endothelial cells were treated with control medium or medium containing10 to 100 ng/ml TNF-α or 300 μg/ml glycated-HAS (treated cells ortreatments) for 24 hrs, in control and compound-added duplicates,containing 10 μM compound. All treatments, compound-added and controlswere carried out in serum free media containing 0.2% albumin. Media fromall conditions were collected and used for IL-6 ELISA.

IL-6 ELISA was carried out using human IL-6 DuoSet ELISA development kitas described by manufacturer (R&D Systems). Mouse anti-human Il-6 wasused as the capture antibody (2 ug/ml) and biotinylated goat anti-humanIL-6 (200 ng/ml) was used as the detection antibody. Culture media wereincubated with capture antibody (in 96 well) for 2 h at roomtemperature. Wells were washed three times with wash buffer (0.05%tween-20 in phosphate buffered saline (PBS) pH 7.4) followed byincubation with detection antibody for 2 h at room temperature.Following three washes wells were incubated with Streptavidin-HRP for 20min. Color development was read at 450 nm in a Microplate reader.

The effects of compounds of the present invention on G-HSA induced IL-6are shown in FIG. 54. G is G-HSA, and C is control, no treatment withcompounds or G-HSA. Endothelial cells under basal conditions secretedabout 25 pg/ml of IL-6. Incubation of endothelial cells with G-HSAinduced a 3 fold increase in IL-6 secretion by endothelial cells.Addition of compounds of the present invention, as indicated by eachcompound's number, to G-HSA containing media significantly reducedendothelial secretion of IL-6. These inhibitory effects varied, the mosteffective compounds showed an 80% decrease in IL-6 secretion. These datashow the compounds of the present invention have anti-inflammatoryactivity.

Example 87 Cytotoxicity/Lactate Dehydrogenase Assay

An appropriate number of cells are plated in four 96-well plates, oneplate for “day 0” and three plates for days 1-3. Cells are treated withat least one compound of the present invention in varying concentrationswith and without the apoptosis inducer cisplatin (2 μM) (“+cis” or“−cis”). Unteated cells are also assayed with and without cisplatin.After transfection, the plates are incubated at 37° C. overnight.

An appropriate number of cells are plated in four 96-well plates, oneplate for “day 0” and three plates for days 1-3. Cells are treated withat least one compound of the present invention in varyingconcentrations. The negative control cells have normal media conditions,a duplicate set of wells is treated with the composition in which thecompound is provided, but there is no added compound and the positivecontrol cells are treated with the apoptosis inducer cisplatin (2 μM).All of the cells are transfected with a vector having a promoter that isresponsive to apoptosis conditions. When apoptosis occurs, the promoteris turned on and the lactic dehydrogenase gene is activated and theenzyme protein is made and active. Activity is easily detected with acolor change. After transfection, the plates are incubated at 37° C.overnight.

About 8 mls of warmed alpha MEM LDH lysis buffer (2% Triton X100) andabout 8 mls of culture media (1/2 dilution) are combined. Two 96-wellv-bottom plates are prepared, one labeled “lysis” and one labeled“supernatant.” To lyse the cells, about 200 μl Alpha MEM lysis buffer(diluted ½) is added to one test plate from which the supernatant hasbeen removed and added to the plate labeled supernatant. After mixing,about 200 μl of lysed cells are transferred to the lysis plate. Both thelysis and supernatant plates are centrifuged at about 1600 rpm for about10 min. After centrifugation, about 100 μl of both the supernatant orlysate is transferred to corresponding 96-well flat-bottomed plates.

The assay for cytotoxicity uses the Cytotoxicity Detection Kit (LDH)from Roche Diagnostics Corp. (Indianapolis, Ind.). Using the directiontsprovided, the dye solution is mixed and added each well of the lysateand supernatant plate and incubated for up to 20-25 min at 15-25° C. inthe dark.

The difference in the amount of lactate dehydrogenase released fromcells in untreated cells when compared to cells treated with cisplatinor compounds of the present invention having cytoxic activity shows thecytotoxic activity of the compounds tested.

Example 88 Synthesis of 4-Benzyloxy-3-chloro-phenylamine (E1)

To 4-amino-2-chlorophenol (7.23 g, 50 mmol) dissolved in acetone (250mL), was added potassium carbonate (6.94 g, 50 mmol), followed by theaddition of benzyl chloride (5.8 mL, 50 mmol), tetrabutylammoniumbromide (TBAB) (1.66 g, 5 mmol), and potassium hydroxide (2.84 g, 50mmol). The reaction mixture was allowed to stir at reflux overnightunder nitrogen. The reaction mixture was extracted three times usingdichloromethane; the combined organic layers were washed with brine anddried over potassium carbonate. The sample was concentrated and theresulting oil was dried overnight under vacuum. The material waspurified by silica gel flash column chromatography eluting with 50:50v:v hexanes:ethyl acetate and the collected fractions were concentratedin vacuo to give E1 (9.3 g, 80%); mp 54° C.; HPLC: Inertsil ODS 3V C18,40:30:30 [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 14.8 min,98.7% purity; MS (TOF ES+) m/z 234 (M+H, 100).

Example 89 Synthesis ofN-(4-Benzyloxy-3-chloro-phenyl)-N′-cycloheptyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine(E2)

To cyanuric chloride (3.148 g, 17.0 mmol) dissolved in acetonitrile (70mL) stirring at −20° C., was added a solution of4-benzyloxy-3-chloro-phenylamine (4.0188 g, 17.0 mmol) in acetonitrile(40 mL) followed by the addition of diisopropylethylamine (DIEA) (3 mL,17.0 mmol), and was stirred at −20° C. for 1 hour under a nitrogenatmosphere. The mixture was allowed to warm to room temperature and thencycloheptylamine (2.2 mL, 17.0 mmol) was added in anhydrous acetonitrile(5 mL) followed by addition of a DIEA (3.2 mL, 18.7 mmol), and themixture was stirred at room temperature overnight. To this reactionmixture was added DIEA (3.2 mL, 18.7 mmol) followed by addition ofN-methyl-4-(methylamino)piperidine (2.5 mL, 17.0 mmol) and was stirredand heated at reflux overnight. The reaction mixture was extracted 3times with methylene chloride; the combined organic layers were washedone time with brine and dried over anhydrous potassium carbonate. Theorganic layer was concentrated in vacuo and dried overnight undervacuum. Column chromatography (90:9:1 v:v:vdichloromethane:methanol:ammonium hydroxide) yielded E2 (619 mg, 7%); mp84° C.; HPLC: Inertsil ODS 3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 37.1 min, 99.4% purity; MS (TOF ES+)m/z 550 (M+H, 100), 276 (M+2H, 34.9), 275.5 (83.9).

Example 90 Synthesis ofN-(4-benzyloxy-3-chloro-phenyl)-N′-cycloheptyl-N″-methyl-N″-piperidin-4-yl-[1,3,5]triazine-2,4,6-triamine(E3)

Compound E3 was obtained as a side product (2.63 g) via columnchromatography (silica gel; 90:9:1 v:v:vdichloromethane:methanol:ammonium hydroxide); mp 74° C.; HPLC: InertsilODS 3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm,R_(t) 38.6 min, 99.8% purity; MS (TOF ES+) m/z 536 (M+H, 100), 269(44.6), 268.5 (M+2H, 97.9).

Example 91 Synthesis of4-[4-Cycloheptylamino-6-(methyl-piperidin-4-yl-amino)-[1,3,5]triazin-2-ylamino]-phenol(E4)

In a dry round bottomed flask was added 10% Pd/C (301.5 mg) and waswetted with 3-5 drops of water. Nitrogen was blown over the Pd/C forabout 5 minutes, then methanol (10 mL) was added cautiously and nitrogenwas blown on the mixture for another 5 minutes. Triazine E3 (251.8 mg,0.50 mmol) dissolved in methanol (5 mL) was added followed by ammoniumformate (381.5 mg, 6.0 mmol), and the reaction was stirred and heatedreflux for about 1.5 hours. Methylene chloride was added and allowed tocool to room temperature. The mixture was filtered by vacuum throughCelite with a methylene chloride rinsing and the filtrate wasconcentrated in vacuo. The material was purified by silica gel flashchromatography eluting with 90:9:1 by volume methylenechloride:methanol:ammonium hydroxide, and the collected fractions weredried over anhydrous magnesium sulfate, filtered and then concentratedin vacuo to give a light yellow solid (E4) (98 mg, 44%); mp 130° C.;HPLC: Inertsil ODS 3V C18, 40:30:30 [KH₂PO₄(0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 3.2 min, 99.6% purity; MS (TOF ES+) m/z412 (M+H, 100), 235.6 (87.2), 206.6 (16.1).

Example 92 Synthesis of4-{4-Cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-1,3,5]triazin-2-ylamino}-phenol(E5)

In a dry flask was added 10% Pd/C (301.0 mg) which was wetted with 3-5drops of water. Nitrogen was blown over the Pd/C for about 5 minutes,then methanol (10 mL) was added cautiously, and nitrogen was blown overthe mixture for another 5 minutes. Triazine E2 (250.5 mg, 0.45 mmol)dissolved in methanol (5 mL) was added followed by ammonium formate(340.5 mg, 5.4 mmol), and the reaction was stirred and heated at refluxfor about 1.5 hours. Methylene chloride was added and allowed to cool toroom temperature. The mixture was filtered by vacuum through Celite witha methylene chloride rinsing and the filtrate was concentrated in vacuo.The crude material was dried overnight under vacuum to give a lightbrown solid (E5) (144 mg, 70%); mp 157° C.; HPLC: Inertsil ODS 3V C18,40:30:30 v:v:v [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 3.0min, 93.8% purity; MS (TOF ES+) m/z 426.3 (M+H, 20.9), 234.1 (100).

Example 93 Synthesis of2-Chloro-4-(4,6-dichloro-[1,3,5]triazin-2-ylamino)-phenol (E6)

Cyanuric chloride (12.95 g, 70.0 mmol) was dissolved in acetone (200mL), the solution was cooled to 0-5° C. with an ice bath, and4-amino-2-chlorophenol (10.09 g, 70.0 mmol) in acetone (100 mL) wasadded. The mixture was stirred at 0-5° C. for 1 hour under nitrogen. Thereaction mixture was extracted three times using dichloromethane; thecombined organic layer were washed with brine and dried over sodiumsulfate. The sample was concentrated in vacuo, and the resulting solidwas dried overnight under vacuum to yield E6 (20.2 g, 99%); mp 180° C.;HPLC: Inertsil ODS 3V C18, 40:30:30 v:v:v [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 11.6 min, 99.4% purity; MS (TOF ES+)m/z 293 (97.5), 291 (M+H, 100).

Example 94 Synthesis of2-Chloro-4-(4-chloro-6-cycloheptylamino-[1,3,5]triazin-2-ylamino)-phenol(E7)

To E7 (1.06 g, 3.4 mmol) dissolved in acetone (25 mL) was added asolution of cycloheptylamine (0.44 mL, 3.4 mmol) in acetone (5 mL)followed by addition of 2.5 N NaOH (1.4 mL, 3.4 mmol) and water (3.5mL). The reaction mixture stirred and heated at reflux for 3 hours. Thereaction mixture was extracted three times using dichloromethane; thecombined organic layers were washed with brine and dried over sodiumsulfate. The sample was concentrated and was dried overnight undervacuum to yield a light brown solid (E7) (1.25 g, 99%); mp 91° C.; HPLC:Inertsil ODS 3V C18, 40:30:30 v:v:v [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 42.9 min, 92.1% purity; MS (TOF ES+)m/z 370 (65.9), 368 (M+H, 100).

Example 95 Alternative Synthesis of2-Chloro-4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-phenol(138)

To E7 (1.00 g, 2.7 mmol) dissolved in THF (25 mL) was added a solutionof N-methyl-4-(methylamino)-piperidine (0.45 mL, 3.1 mmol) in THF (5 mL)followed by addition of 2.5 N NaOH (1.1 mL, 2.7 mmol) and 2.5 mL ofwater. The reaction mixture was stirred and heated at reflux overnight.The reaction mixture was extracted three times using dichloromethane,the combined organic layers were washed with a brine and dried oversodium sulfate. The sample was concentrated and the resulting solid wasdried overnight under vacuum. Column chromatography (silica gel, 100%methanol) yielded an off-white solid (138) (177 mg, 14%); mp 68° C.;HPLC: Inertsil ODS 3V C18, 40:30:30 v:v:v [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 4.7 min, 99.6% purity; MS (TOF ES+) m/z460 (M+H, 55.3), 251 (100), 224 (51.1).

Example 96 Synthesis ofN-(1-Benzyl-piperidin-4-yl)-N′-(3-fluoro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-diamine(E8)

To 127 (6.00 g, 16.4 mmol) dissolved in THF (100 mL) was added4-amino-1-benzylpiperidine (3.6 mL, 18.9 mmol) in THF (5 mL), followedby addition of H₂O (17 mL) and 2.5 N NaOH (6.6 mL, 16.4 mmol). Thereaction mixture was stirred and heated at reflux for about 12 hoursunder nitrogen. The reaction mixture was extracted three times withdichloromethane; the combined organic layers were washed with brine anddried over potassium carbonate. The sample was filtered, concentrated,and the resulting solid was dried overnight under vacuum. Columnchromatography (93:6:1 v:v:v dichloromethane:methanol:ammoniumhydroxide) gave a light yellow solid (E8) (2.98 g, 35%); mp 87° C.;HPLC: Inertsil ODS 3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 11.1 min, 93.9% purity; MS (TOF ES+)m/z 520 (M+H, 66.3), 430 (100).

Example 97 Alternative Synthesis ofN-(1-Benzyl-piperidin-4-yl)-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-diamine(134)

To 133 (6.04 g, 15.7 mmol) dissolved in THF (100 mL) was added4-amino-1-benzylpiperidine (3.4 mL, 18 mmol) in THF (5 mL), followed byaddition of water (16 mL) and 2.5 N NaOH (6.3 mL, 15.7 mmol). Thereaction mixture was stirred and heated at reflux for about 12 hoursunder a nitrogen atmosphere. The reaction mixture was extracted threetimes with dichloromethane; the combined organic layers were washed withbrine and dried over potassium carbonate. The sample was filtered,concentrated, and the resulting solid was dried overnight under vacuum.Column chromatography (93:6:1 v:v:v dichloromethane:methanol:ammoniumhydroxide) gave a light yellow solid (134) (3.71 g, 44%). mp 90° C.;HPLC: Inertsil ODS 3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 14.6 min, 99.5% purity; MS (TOF ES+)m/z 538 (27.8), 536 (M+H, 72.1), 448 (39.2), 446 (100).

Example 98 Synthesis ofN-Cycloheptyl-N′-(4-methoxy-phenyl)-N″-piperidin-4-yl-[1,3,5]triazine-2,4,6-triamine(E9)

In a dry round bottomed flask, 10% Pd/C (600.2 mg) was added and waswetted with 3-5 drops of water. Nitrogen was blown over the Pd/C forabout 5 minutes, then methanol (25 mL) was added cautiously, andnitrogen was again blown over the mixture for about 5 minutes. Triazine134 (501.2 mg, 0.93 mmol) dissolved in methanol (15 mL) was addedfollowed by ammonium formate (708.4 mg, 11.2 mmol), and the reaction wasstirred and heated at reflux for about 1.5 hours. Methylene chloride wasadded and allowed to cool to room temperature. The mixture was filteredby vacuum through Celite with a methylene chloride rinsing, the filtratewas concentrated, and the material was dried overnight under vacuum.Column chromatography (90:9:1 v:v:v dichloromethane:methanol:ammoniumhydroxide) yielded a light yellow solid (E9) (226 mg, 55%); mp 118° C.;HPLC: Inertsil ODS 3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 4.6 min, 99.6% purity; MS (TOF ES+) m/z412 (M+H, 39.8), 247.9 (63), 227.3 (100).

Example 99 Synthesis of6-Chloro-N-cyclopropyl-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine(E10)

To 124 (2.01 g, 7.0 mmol) dissolved in acetone (40 mL) was added asolution of cyclopropylamine (0.5 mL, 7 mmol) in acetone (5 mL) followedby addition of NaOH (2.8 mL, 2.5 N, 7.0 mmol) and 8 mL of water. Thereaction was stirred and heated at reflux for 3 hours. The reactionmixture was poured over crushed ice. The solid that formed was collectedby vacuum filtration and was dried overnight under vacuum to affordunpurified E10 (1.8 g). {A separate reaction [124, (2.00 g, 7 mmol) andcyclopropylamine (0.5 mL, 7 mmol)] yielded unpurified E10 (1.9 g).} Thetwo lots (1.8 g and 1.9 g) were combined for purification. Columnchromatography (50:50 v:v hexane:ethyl acetate) yielded a faint yellowsolid (E10) (2.89 g, 67%); mp 186° C.; HPLC: Inertsil ODS 3V C18,40:30:30 [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 16.6 min,94.9% purity; MS (TOF ES+) m/z 310 (M+H, 100), 312 (44.4).

Example 100 Synthesis ofN-Cyclopropyl-N′-(3-flouro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine(E11)

To E10 (1.04 g, 3.2 mmol) dissolved in THF (40 mL) was added a solutionof N-methyl-4-(methyl-amino)-piperidine (0.5 mL, 3.2 mmol) in THF (2 mL)followed by addition of NaOH (1.3 mL, 2.5 N, 3.2 mmol) and 3.5 mL ofwater. The reaction mixture stirred and heated at reflux for 2 hours.The reaction mixture was extracted 3 times using dichloromethane; thecombined organic layers were washed with brine and dried over potassiumcarbonate. The sample was filtered, concentrated, and the resultingsolid was dried overnight under vacuum. Column chromatography (90:9:1v:v:v dichloromethane:methanol:ammonium hydroxide) yielded a lightyellow solid (E11) (164 mg, 13%); mp 94° C.; HPLC: Inertsil ODS 3V C18,40:30:30 [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 3.2 min,96.7% purity; MS (TOF ES+) m/z 402.1 (M+H, 100), 231 (41.5), 202.1 (6).

Example 101 Synthesis ofN-Cyclopropyl-N′-(1-ethyl-pyrrolidin-2-ylmethyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine(E12)

To E10 (1.05 g, 3.2 mmol) dissolved in THF (40 mL) was added a solutionof 2-(aminomethyl)-1-ethylpyrrolidine (0.5 mL, 3.2 mmol) in THF (2 mL)followed by addition of NaOH (1.3 mL, 2.5 N, 3.2 mmol) and 3.5 mL ofwater. The reaction mixture was stirred and heated at reflux for 2hours. The reaction mixture was extracted 3 times using dichloromethane;combined organic layers were washed with brine and dried over potassiumcarbonate. The sample was filtered, concentrated, and dried overnightunder vacuum. Column chromatography (90:9:1 v:v:vdichloromethane:methanol:ammonium hydroxide) yielded a light yellowsolid (E12) (755 mg, 59%); mp 68° C.; HPLC: Inertsil ODS 3V C18,40:30:30 [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 3.2 min,95.5% purity; MS (TOF ES+) m/z 402.1 (M+H, 100), 231. (23.4).

Example 102 Synthesis of6-Chloro-N-(3-chloro-4-methoxy-phenyl)-N′-cyclopropyl-[1,3,5]triazine-2,4-diamine(E13)

To 101 (2.01 g, 6.5 mmol) dissolved in acetone (35 mL) was added asolution of cyclopropylamine (0.45 mL, 6.5 mmol) in acetone (5 mL)followed by addition of NaOH (2.6 mL, 2.5 N, 7.0 mmol) and 6.5 mL ofwater. The reaction mixture was stirred and heated at reflux for 3hours. The reaction mixture was extracted 3 times with dichloromethane;the combined organic layers were washed with brine and dried overpotassium carbonate. The sample was filtered, concentrated, and driedovernight under vacuum. Column chromatography (50:50 v:v hexane:ethylacetate) yielded a faint yellow solid (E13) (1.12 g, 53%); mp 172° C.;HPLC: Inertsil ODS 3V C18, 40:30:30 [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 24.6 min, 99% purity; MS (TOF ES+) m/z328 (72.6), 326 (M+H, 100).

Example 103 Synthesis ofN-Cyclopropyl-N′-(3-chloro-4-methoxy-phenyl)-N″-methyl-″N-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine(E14)

To E13 (0.88 g, 2.7 mmol) dissolved in THF (40 mL) was added a solutionof N-methyl-4-(methyl-amino)-piperidine (0.4 mL, 2.7 mmol) in THF (2 mL)followed by addition of NaOH (1.1 mL, 2.5 N, 2.7 mmol) and 3 mL ofwater. The reaction mixture was stirred and heated at reflux for 2hours. The reaction mixture was extracted 3 times with dichloromethane;the combined organic layers were washed with brine and dried overpotassium carbonate. The sample was filtered, concentrated, and theresulting solid was dried overnight under vacuum. Column chromatography(90:9:1 v:v:v dichloromethane:methanol:ammonium hydroxide) yielded alight yellow solid (E14) (93 mg, 8.3%); mp 92° C.; HPLC: Inertsil ODS 3VC18, 40:30:30 [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 3.4min, 96.9% purity; MS (TOF ES+) m/z 418.1 (M+H, 100), 210.1 (12.3).

Example 104 Synthesis of6-Chloro-N,N′-bis-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine(E15)

To 124 (1.09 g, 3.5 mmol) dissolved in acetone (25 mL) was added3-fluoro-p-anisidine (0.54 g, 3.5 mmol) in acetone (5 mL) followed byaddition of 2.5 N NaOH (1.4 mL, 3.5 mmol) and water (3.5 mL). Thereaction mixture was stirred and heated at reflux for 3 hours. Thereaction mixture was extracted three times using dichloromethane; thecombined organic layers were washed with brine and dried over potassiumcarbonate. The sample was filtered, concentrated in vacuo, and theresulting solid was dried overnight under vacuum to yield white solid(E15) (1.332 g, 97%); mp 194° C.; HPLC: Inertsil ODS 3V C18, 40:30:30v:v:v [KH₂PO₄ (0.01 M, pH 3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 33.2 min,97.6% purity; MS (TOF ES+) m/z 396 (35.0); 394 (M+H, 100).

Example 105 Synthesis ofN-(1-Ethyl-pyrrolidin-2-ylmethyl)-N′,N″-bis-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine(E16)

To E15 (1.01 g, 2.5 mmol) dissolved in THF (25 mL) was added a solutionof 2-(aminomethyl)-ethyl pyrrolidine (0.42 mL, 2.9 mmol) in THF (5 mL)followed by addition of 2.5 N NaOH (1.0 mL, 2.5 mmol) and 2.5 mL ofwater. The reaction mixture was stirred and heated at reflux overnight.The reaction mixture was extracted three times using dichloromethane;the combined organic layers were washed with brine and dried overpotassium carbonate. The sample was filtered, concentrated in vacuo, andthe resulting solid was dried overnight under vacuum. Columnchromatography (90:9:1 v:v:v dichloromethane:methanol:ammoniumhydroxide) yielded a light yellow solid (E16) (570 mg, 47%); mp 66° C.;HPLC: Inertsil ODS 3V C18, 40:30:30 v:v:v [KH₂PO₄ (0.01 M, pH3.2):CH₃OH:CH₃CN], 264 nm, R_(t) 4.7 min, 99.3% purity; MS (TOF ES+) m/z486 (68.4, M+H), 264 (100), 244 [(M+2H)++, 48.1).

Example 106 Synthesis of 1-[4-(3-chloro-4-methoxyanilino)-cycloheptylamino-1,3,5-triazin-2-yl]-2-azoloamymethanol (E17)

To 133 (0.2 g, 0.52 mmol) in Dowtherm (5 mL) was added slowly prolinol(0.16 g, 1.57 mmol) and the mixture was heated to 150-160° C. for 5hours with stirring. After completion of the reaction the mixture wascooled to 25° C. and then transferred directly to a silica gel columnfor purification purpose. The column was eluted using 1:1 (by volume)EtOAc-Petroleum ether to afford E17 as a light brown solid (0.14 g,60%), mp 82-84° C.; HPLC: Inertsil ODS 3V (250×4.6 mm) 5 microns 50:50[0.01 M KH₂PO₄ (0.1% TFA):CH₃CN], 223 nm, R_(t) 11.86 min, 97.49%purity); MS (CI): m/z 447 (M⁺, 100), 416 (35).

Example 107 Synthesis ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(4-methylpiperzino)-1,3,5-triazine-4,2-diamine(E18)

To a solution of 133 (0.5 g, 1.31 mmol) in 1,4-dioxane (5 mL) was addedslowly a solution of 1-methylpiperizine (0.13 g, 1.31 mmol) in1,4-dioxane (5 mL) followed by 2.5 N sodium hydroxide solution (0.5 mL,1.31 mmol) and water (1.2 mL). The mixture was heated to reflux for2.5-3 hours with stirring under nitrogen atmosphere and then cooled to25° C. and diluted with water (10 mL). The mixture was then stirred for10-15 min., the precipitated solid was filtered off and dried undervacuum to afford the title compound E18 as an off-white solid (0.5 g,86%). mp 120-122° C.; HPLC: Inertsil ODS 3V (250×4.6 mm) 5 microns 65:35[0.01 M KH₂PO₄ (0.1% TFA):CH₃CN], 223 nm, R_(t) 10.23 min, 98.50%purity); MS (CI): m/z 446 (M+H, 100), 375 (25).

Example 108 Synthesis of3-[4-(3-chloro-4-methoxyanilino)-6-cycloheptylamino-1,3,5-triazin-2-yloxy]-2-ethyl-4H-4-pyranone(E19)

A mixture of 133 (0.3 g, 0.78 mmol), 2-ethyl-3-hydroxy-4H-pyran-4-one(0.11 g, 0.78 mmol) and K₂CO₃ (0.54 g, 3.91 mmol) in dimethylformamide(15 mL) was stirred at 80° C. for 6 hours under nitrogen atmosphere.After completion of the reaction the mixture was cooled to 25° C. anddiluted with water (50 mL). The precipitated solid was filtered off anddried under vacuum to afford the title compound E19 as an off-whitesolid (0.2 g, 52%). mp 124-126° C.; HPLC: Inertsil ODS 3V (250×4.6 mm) 5microns 50:50 [0.01 M KH₂PO₄ (0.1% TFA):CH₃CN], 223 nm, R_(t) 18.47 min,98.18% purity); MS (CI): m/z 486 (M⁺, 100), 382 (90).

Example 109 Synthesis of1-[3-{4-(3-chloro-4-methoxyanilino)-6-cycloheptylamino-1,3,5-triazine-2-yloxy}piperidino]-1-ethanone(E20)

A mixture of N-acetyl-3-hydroxypiperidine (0.335 g, 2.27 mmol) andsodium hydroxide (95 mg, 2.27 mmol) in benzene (10 mL) was heated toreflux for 2 hours with stirring under nitrogen atmosphere and thencooled to 25° C. followed by the addition of compound 133 (0.3 g, 0.78mmol) at same temperature. The mixture was heated to reflux for 6 hours,concentrated under vacuum and diluted with water (10 mL). Theprecipitated solid was filtered off and purified by columnchromatography to afford the title compound E20 as an off-white solid(0.35 g, 91%). mp 138-140° C.; HPLC: Inertsil ODS 3V (250×4.6 mm) 5microns [solvent A=0.01 M KH₂PO₄ (pH 7.0); solvent B=CH₃CN], Gradientelution program: T/% B=0/60, 10/60, 25/80, 40/80, 45/60, 50/60; 268 nm,R_(t) 17.20 min, 91.70% purity; MS (CI): m/z 489 (M⁺, 100).

Example 110 Synthesis ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-isopropoxy-1,3,5-triazine-2,4-diamine(E21)

A mixture of isopropanol (2 mL) and sodium hydroxide (95 mg, 2.27 mmol)in benzene (10 mL) was heated to reflux for 2 hours with stirring undernitrogen atmosphere and then cooled to 25° C. followed by the additionof compound 133 (0.3 g, 0.78 mmol) at same temperature. The mixture washeated to reflux for 6 hours, concentrated under vacuum and diluted withwater (10 mL). The precipitated solid was filtered off and purified bycolumn chromatography (1-2% MeOH—CHCl₃) to afford the title compound E21as an off-white solid (0.30 g, 94%). mp 136-138° C.; HPLC: Inertsil ODS3V (250×4.6 mm) 5 microns [solvent A=0.01 M KH₂PO₄ (pH 7.0); solventB=CH₃CN], Gradient elution program: T/% B=0/60, 10/60, 25/80, 40/80,45/60, 50/60; 268 nm, R_(t) 30.06 min, 98.78% purity; MS (CI): m/z 406(M+H, 100).

Example 111 Synthesis ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(2-azolanylmethoxy)-1,3,5-triazine-2,4-diamine(E22)

A mixture of prolinol (0.0243 g, 2.27 mmol) and sodium hydroxide (95 mg,2.27 mmol) in benzene (10 mL) was heated to reflux for 2 hours withstirring under nitrogen atmosphere and then cooled to 25° C. followed bythe addition of compound 133 (0.3 g, 0.78 mmol) at same temperature. Themixture was heated to reflux for 6 hours, concentrated under vacuum anddiluted with water (10 mL). The precipitated solid was filtered off andpurified by column chromatography (1-2% MeOH—CHCl₃) to afford the titlecompound E22 as an off-white solid (0.35 g, quantitative). mp 84-86° C.;HPLC: Inertsil ODS 3V (250×4.6 mm) 5 microns [solvent A=0.01 M KH₂PO₄(pH 7.0); solvent B=CH₃CN], Gradient elution program: T/% B=0/60, 10/60,25/80, 40/80, 45/60, 50/60; 268 nm, R_(t) 24.67 min, 99.59% purity; MS(CI): m/z 447 (M+H, 100).

Example 112 Synthesis of1-[4-(3-chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazine-2-yl]-piperidin-3-ol](E23)

A mixture of 3-hydroxypiperidine (0.198 g, 1.96 mmol) and sodiumhydroxide (79 mg, 1.96 mmol) in benzene (10 mL) was heated to reflux for2 hours with stirring under nitrogen atmosphere and then cooled to 25°C. followed by the addition of compound 133 (0.25 g, 0.65 mmol) at sametemperature. The mixture was heated to reflux for 6 hours, concentratedunder vacuum and diluted with water (10 mL). The precipitated solid wasfiltered off and purified by column chromatography (1-2% MeOH—CHCl₃) toafford the title compound E23 as an off-white solid (0.06 g, 21%). mp100-102° C.; HPLC: Hichrom RPB (250×4.6 mm) 5 microns [solvent A=0.01 MKH₂PO₄ (pH 5.5); solvent B=CH₃CN], Gradient elution program: T/% B=0/20,10/20, 25/80, 40/80, 55/60, 65/20, 70/20; 270 nm, R_(t) 41.95 min,98.43% purity; MS (CI): m/z 447 (M+H, 100).

Example 113 Synthesis of1-[4-(3-chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazine-2-yl]-piperidin-4-ol](E24)

A mixture of 4-hydroxypiperidine (0.198 g, 1.96 mmol) and sodiumhydroxide (79 mg, 1.96 mmol) in benzene (10 mL) was heated to reflux for2 hours with stirring under nitrogen atmosphere and then cooled to 25°C. followed by the addition of compound 133 (0.25 g, 0.65 mmol) at sametemperature. The mixture was heated to reflux for 6 hours, concentratedunder vacuum and diluted with water (10 mL). The precipitated solid wasfiltered off and purified by column chromatography (1-2% MeOH—CHCl₃) toafford the title compound E24 as an off-white solid (0.20 g, 58%). mp142-144° C.; HPLC: Hichrom RPB (250×4.6 mm) 5 microns [solvent A=0.01 MKH₂PO₄ (pH 5.5); solvent B=CH₃CN], Gradient elution program: T/% B=0/20,10/20, 25/80, 40/80, 55/60, 65/20, 70/20; 270 nm, R_(t) 40.56 min,98.60% purity; MS (CI): m/z 447 (M+H, 100).

Example 114 Synthesis ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(1-methyl-2-azolanylmethoxy)-1,3,5-triazine-2,4-diamine(E25)

A mixture of N-methyl prolinol (0.226 g, 1.96 mmol) and sodium hydroxide(79 mg, 1.96 mmol) in benzene (10 mL) was heated to reflux for 2 hourswith stirring under nitrogen atmosphere and then cooled to 25° C.followed by the addition of compound 133 (0.25 g, 0.65 mmol) at sametemperature. The mixture was heated to reflux for 6 hours, concentratedunder vacuum and diluted with water (3 mL). The gum which separated wascollected (by decanting out the liquid portion) and purified by columnchromatography (1-3% MeOH—CHCl₃) to afford the title compound E25 as asemi-solid (0.06 g, 20%). HPLC: Symmetry shield RP18 (250×4.6 mm) 5microns [solvent A=0.01 M KH₂PO₄ (pH 3.0); solvent B=CH₃CN], Gradientelution program: T/% B=0/35, 10/35, 40/80, 50/80, 55/35, 60/35; 270 nm,R_(t) 15.51 min, 99.55% purity; MS (CI): m/z 461 (M+H, 100).

Example 115 Synthesis ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(1-methyl-4-piperidyloxy)-1,3,5-triazine-2,4-diamine(E26)

A mixture of N-methyl piperidinol (0.22 g, 1.96 mmol) and sodiumhydroxide (79 mg, 1.96 mmol) in benzene (10 mL) was heated to reflux for2 hours with stirring under nitrogen atmosphere and then cooled to 25°C. followed by the addition of compound 133 (0.25 g, 0.65 mmol) at sametemperature. The mixture was heated to reflux for 6 hours, concentratedunder vacuum and diluted with water (10 mL). The precipitated solid wasfiltered and purified by column chromatography (5-10% MeOH—CHCl₃) toafford the title compound E26 as a pale yellow solid (0.16 g, 53%).HPLC: Hichrom RPB (250×4.6 mm) 5 microns [solvent A=0.01 M KH₂PO₄ (pH7.0); solvent B=CH₃CN], Gradient elution program: T/% B=0/60, 10/60,25/80, 40/80, 45/60, 50/60; 270 nm, R_(t) 35.21 min, 98.09% purity; MS(CI): m/z 461 (M+H, 50), 364 (100).

Example 116 Synthesis ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(1,4-thiazinan-4-yl)-1,3,5-triazine-4,2-diamine(E27)

A mixture of thiomorpholine (97 mg, 0.942 mmol), sodium hydroxide (31.4mg, 0.785 mmol) and water (0.5 mL) in 1,4-dioxane (15 mL) was heated toreflux for 3 hours with stirring under nitrogen atmosphere and thencooled to 25° C. followed by the addition of compound 133 (0.30 g, 0.785mmol) at same temperature. The mixture was heated to reflux for 12hours, concentrated under vacuum and diluted with water (20 mL). Themixture was then extracted with EtOAc (2×20 mL). The combined organiclayers were washed with water (2×10 mL), dried over anhydrous Na₂SO₄ andconcentrated under vacuum. The residue thus obtained was purified bycolumn chromatography (10-20% EtOAc-Hexane) to afford the title compoundE27 as a pale yellow solid (0.26 g, 74%). mp 78-80° C.; HPLC: InertsilODS 3V (250×4.6 mm) 5 microns [solvent A=0.01 M KH₂PO₄; solventB=CH₃CN], A:B=20:80; 220 nm, R_(t) 20.36 min, 97.15% purity; MS (CI):m/z 449 (M+H, 100).

Example 117 Synthesis ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(2-fluorophenoxy)-1,3,5-triazine-4,2-diamine(E28)

A mixture of compound 133 (0.3 g, 0.785 mmol), 2-fluorophenol (0.106 g,0.942 mmol) and potassium carbonate (541 mg, 3.93 mmol) indimethylformamide (3 mL) was heated to 80° C. for 12 hours with stirringunder nitrogen atmosphere. The mixture was then cooled, diluted withwater (20 mL) and extracted with EtOAc (2×20 mL). The combined organiclayers were washed with water (2×10 mL), dried over anhydrous sodiumsulphate and concentrated under vacuum. The residue thus obtained waspurified by column chromatography (10-20% EtOAc-Hexane) to afford thetitle compound E28 as a pale yellow solid (0.2 g, 56%). mp 98-100° C.;HPLC: Inertsil ODS 3V (250×4.6 mm) 5 microns [solvent A=0.01 M KH₂PO₄(pH 7.0); solvent B=CH₃CN], Gradient elution program: T/% B=0/60, 10/60,25/80, 40/80, 45/60, 50/60; 268 nm, R_(t) 29.79 min, 99.82% purity; MS(CI): m/z 458 (M+H, 100).

Example 118 Synthesis ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-[2-(2-fluorophenoxy)ethoxy]-1,3,5-triazine-4,2-diamine(E29)

A mixture of 2-(2-fluorophenoxy)-1-ethanol (367 mg, 2.356 mmol) andsodium hydroxide (94 mg, 2.356 mmol) in benzene (15 mL) was heated toreflux for 3 hours with stirring under nitrogen atmosphere and thencooled to 25° C. followed by the addition of compound 1 (0.30 g, 0.785mmol) at same temperature. The mixture was heated to reflux for 12hours, concentrated under vacuum and diluted with water (20 mL). Themixture was then extracted with EtOAc (2×20 mL). The combined organiclayers were washed with water (2×10 mL), dried over anhydrous sodiumsulphate and concentrated under vacuum. The residue thus obtained waspurified by column chromatography (10-20% EtOAc-Hexane) to afford thetitle compound E29 as a semi solid (90 mg, 23%). HPLC: Inertsil ODS 3V(250×4.6 mm) 5 microns [solvent A=0.01 M KH₂PO₄ (pH 7.0); solventB=CH₃CN], Gradient elution program: T/% B=0/60, 10/60, 25/80, 40/80,45/60, 50/60; 268 nm, R_(t) 32.31 min, 99.30% purity; MS (CI): m/z 502(M+H, 100).

Example 119 Synthesis ofN²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(6-methyl-2-pyrylmethoxy]-1,3,5-triazine-4,2-diamine(E30)

A mixture of 6-methyl-2-pyridylmethanol (145 mg, 1.17 mmol) and sodiumhydroxide (63 mg, 1.57 mmol) in benzene (15 mL) was heated to reflux for3 hours with stirring under nitrogen atmosphere and then cooled to 25°C. followed by the addition of compound 133 (0.30 g, 0.785 mmol) at sametemperature. The mixture was heated to reflux for 12 hours, concentratedunder vacuum and diluted with water (20 mL). The mixture was thenextracted with EtOAc (2×20 mL). The combined organic layers were washedwith water (2×10 mL), dried over anhydrous sodium sulphate andconcentrated under vacuum. The residue thus obtained was purified bycolumn chromatography (70-80% EtOAc-Hexane) to afford the title compoundE30 as a hygroscopic solid (257 mg, 70%). HPLC: Inertsil ODS 3V (250×4.6mm) 5 microns [solvent A=0.01 M KH₂PO₄ (pH 7.0); solvent B=CH₃CN],Gradient elution program: T/% B=0/60, 10/60, 25/80, 40/80, 45/60, 50/60;268 nm, R_(t) 23.93 min, 99.14% purity; MS (CI): m/z 469 (M⁺, 100).

Example 120 Synthesis ofN-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methylpiperidin-4-yl)[1,3,5]triazine-2,4,6-triamine (137)

N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methylpiperidin-4-yl)[1,3,5]triazine-2,4,6-triamine (137) is a 1,3,5-triazinederivative which is synthesized from 2,4,6-trichloro-1,3,5-triazine(cyanuric chloride) by serial substitution of amines, for example,3-chloro-4-methoxyaniline, cycloheptylamine, and1-methyl-(4-methylamino)piperidine, respectively.

This reaction scheme illustrates synthesis of compound 137 by reactionof cyanuric chloride with 3-chloro-4-methoxyaniline using sodiumhydroxide (NaOH) as base at 0-5° C. to yield (3-chloro-4-methoxyphenyl)-(4,6-dichloro-[1,3,5]triazine-2-yl) amine (compound 101), whichon reaction with cycloheptylamine in presence of NaOH under reflux gave6-chloro-N-(3-Chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine(compound 133). Compound 133 on reaction with1-methyl-(4-methylamino)piperidine yields, the final compound 137.

Example 121 Alternative Synthesis ofN-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methylpiperidin-4-yl)[1,3,5]triazine-2,4,6-triamine (137)

A 1 kg campaign was conducted to develop a process which avoids multiplecolumn purifications and employs a suitable recrystallisation method toproduce compound 137. This scheme was stabilized for the campaign,however the scale up of compound 133 by one pot synthesis resulted inthe formation of an impurity, which was carried over to the final stepand was difficult to remove. The impurity was isolated and characterizedto be6-chloro-N-(3-chloro-4-methoxyphenyl)-N′-(3-chloro-4-ethoxyphenyl)[1,3,5]triazine-2,4-diamine.To avoid the impurity, the single pot reaction was avoided and compound101 was isolated and purified by recrystallisation in ethylacetate.

Attempts to minimize the impurity involved scanning several bases andsolvents, but the production6-chloro-N-(3-chloro-4-methoxyphenyl)-N′-(3-chloro-4-methoxyphenyl)[1,3,5]triazine-2,4-diamineremained. The preparation of compound 101 was then carried out withoutaddition of base, resulting in the levels of the impurity beingnegligible. Compound 133 was prepared from compound 101 using aqueousNaOH as base.

Example 122 General Large Scale Synthesis ofN-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methylpiperidin-4-yl)[1,3,5]triazine-2,4,6-triamine (137)

Among the bases and solvents screened, sodium acetate (NaOAc) and1,4-dioxane showed consistently good results for a 1 kg preparation.This scheme involves the preparation of compound 101 by reaction ofcyanuric chloride with 3-chloro-4-methoxyaniline in acetone at 0-5° C.in the absence of base, which is then made to react withcycloheptylamine in presence of NaOH under reflux to give compound 133.This compound 133 was reacted with 1-methyl-(4-methylamino)piperidineusing NaOAc in 1,4-dioxane solvent to yield the crude compound 137 whichis purified by making hydrochloride salt,N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methylpiperidin-4-yl)[1,3,5]triazine-2,4,6-triamine, HCl salt, compound E31and by recrystallisation in isopropanol. The hydrochloride salt was thenreleased to give the free base, compound 137. Compound obtained in thismanner exhibited the same high quality of appearance, purity, lowresidual solvents, assay and impurity profile, and the like.

Example 123 Large Scale Synthesis of(3-chloro-4-methoxyphenyl)-(4,6-dichloro-[1,3,5]triazine-2-yl)amine(101)

As provided in the reaction scheme above, a solution of3-chloro-4-methoxyaniline (42.5 g) in acetone was slowly added to thecyanuric chloride solution (50 g dissolved in acetone at −10-0° C. Thereaction mass was maintained at the same temperature for about 1 hourafter addition. The progress of the reaction was monitored by TLC. Afterthe completion of the reaction, the reaction mass was poured overcrushed ice under stirring. The solid compound thrown out was filteredunder vacuum, washed thoroughly with water and then dried. The abovecompound was recrystallised from ethylacetate (Wt. 60 g Y: 73%).

The following Table illustrates the results obtained using variousbases, solvents, and other reaction conditions.

Rxn Yield Temp. Cmpdd Disub Trisubstit No. Base Solvent (%) ° C.Purity(%) (%) (%) Remarks 1 NaOH Acetone 92 0–5 90 4–6 1–2 2 NaOHAcetone 75  5–10 86 7–8 2–3 3 NaOH Acetone 80 0–5 40 35 20 Reverseaddition 4 NaOH Acetone 85 30 65 15–20 5–8 5 NaOH Acetone 89 Reflux 2060 10–15 6 K₂CO₃ Acetone 90 0–5 91 7–8 1–2 7 K₂CO₃ Toluene 90 0–5 91 7–81–2 8 NaOH Toluene 80 0–5 85 6–7 1–2 9 NaOH Dioxane 85 0–5 92 5–6 — 10K₂CO₃ Dioxane 90 0–5 91 5–6 — 11 K₂CO₃ Dioxane 85 0–5 55 20–25 4–5Reverse addition 12 — Acetone 95 0–5 95 1–2 — 13 — Acetone 98 <0 99 — —14 Basic Acetone 85 0–5 95 — — resin

Example 124 Large Scale Synthesis of6-chloro-N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine(133)

As provided in the reaction scheme above, a solution of cycloheptylamine(20.4 g) in acetone was slowly added to the 101 solution (50 g dissolvedin acetone) at 20-30° C. To this stirring solution water (100 ml) wasadded followed by Aq. sodium hydroxide solution (6.5 g dissolved inwater). Then the temperature of the reaction mass raised to reflux andmaintained for about 2-3 h. TLC was monitored. After completion of thereaction, temperature of the RM (reaction mixture) was brought to 25-30°C. and the reaction mass was poured over chilled water under stirring.The solid compound thrown out was filtered and dried (Wt. 60 g Y:95.8%).

The following Table illustrates the results obtained using variousbases, solvents, and other reaction conditions.

Rxn Yield Cmpd No. Base Solvent (%) Purity(%) Remarks 1 NaOH Acetone 9295 2 NaOH Toluene 80 90 3 NaOH Dioxane 80 82 4 K₂CO₃ Acetone 95 99 5K₂CO₃ Dioxane 89 85 6 Et₃N Acetone 85 75 7 Et₃N Dioxane 75 60 8 Et₃NToluene 70 65

Example 125 One-Pot Synthesis of6-chloro-N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine(133)

A convenient, one-pot synthesis of6-chloro-N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diaminewas carried out as follows. To cyanuric chloride (50 g) dissolved inacetone was added a solution of 3-chloro-4-methoxyaniline (42.5 g) inacetone at 0-5° C. followed by the addition of potassium carbonate (112g). The reaction mass was maintained at the same temperature for about 1hour. The progress of the reaction was monitored by TLC. When thereaction was complete, cycloheptylamine (29 g dissolved in acetone) wasadded at 0-10° C. The reaction mass was maintained under reflux forabout 2-3 hours. Completion of the reaction was checked by TLC. Thereaction mass was cooled to 20-30° C. and poured over crushed ice understirring. The solid compound thrown out was filtered and dried. Thecrude compound was then recrystallised from ethylacetate (Wt. 50 g Y:48%).

Example 126 Large-Scale Synthesis ofN-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methylpiperidin-4-yl)[1,3,5]triazine-2,4,6-triamine (137)

1-Methyl-(4-methylamino)piperidine (17 g in 1,4-dioxane) was added tothe solution of6-chloro-N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine(50 g dissolved in 1,4-dioxane) at RT followed by addition of sodiumacetate (21.5 g). The RM (reaction mixture) was maintained under refluxfor about 2-3 hours. The progress of the reaction was monitored by TLC.After the completion of the reaction, the reaction mass was cooled to20-30° C. and filtered through hi-flow bed. The filtrate was evaporatedto minimum volume and was diluted with toluene. This was acidified withdilute HCl under stirring conditions. The solid obtained was filteredand dried. The above crude compound was recrystallised from isopropanol(Wt. 20 g Y: 30%).

The HCl salt (20 g) was dissolved in methanol, diluted with ethylacetateand made basic with aqueous potassium carbonate solution. The organiclayer was separated and the aqueous layer was again extracted intoethylacetate. The organic layers were combined, washed with water andevaporated to yield the compound (Wt. 20 g Y: 30%).

Example 127 General Synthesis of Akoxide Derivatives of6-chloro-N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine

The sequential substitution at the triazine core permitted theconvenient formation of alkoxide derivatives of triazines. The procedureinvolves the preparation of compound 101 by reaction of cyanuricchloride with 3-chloro-4-methoxyaniline in acetone at 0-5° C. in theabsence of base, which is then made to react with cycloheptylamine inpresence of sodium hydroxide under reflux to give compound 133. Thiscompound 133 was refluxed in alcohols like methanol, ethanol,isopropanol, etc., with base to yield alkoxide compounds whosestructures are confirmed by the spectral data.

Example 128 Synthesis of 6-methoxy-N-(3-Chloro-4-methoxyphenyl)-N′-cyclo heptyl-[1,3,5]triazine-2,4 diamine (E32)

To 6-chloro-N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine (5 g) dissolved in methanol was added K₂CO₃ (3.2 g)and refluxed for about 6-8 h. TLC was monitored. After the completion ofthe reaction, the reaction mass was filtered through vacuum and thefiltrate was evaporated to yield the title compound. (Wt. 4.7 g Y: 95%)

Example 129 Synthesis of 6-ethoxy-N-(3-Chloro-4-methoxy phenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4 diamine (E33)

A portion of6-chloro-N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine(5 g) was refluxed in ethanol in presence of K₂CO₃ (3.2 g) for about8-10 h. TLC was monitored. After the completion of the reaction, thereaction mass was filtered through vacuum and the filtrate wasevaporated to yield the title compound. (Wt. 4.6 g Y: 90%).

Example 130 Synthesis of 6-isopropoxy-N-(3-Chloro-4-methoxyphenyl)-N′-cyclo heptyl-[1,3,5]triazine-2,4 diamine (E34)

To a portion of6-chloro-N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine(5 g) in isopropanol was added K₂CO₃ (3.2 g) and refluxed for about10-12 h. TLC was monitored. After the completion of the reaction, thereaction mass was filtered through vacuum and the filtrate wasevaporated to yield the title compound. (Wt. 3.7 g Y: 70%).

Example 131 Alternative Synthesis of 6-alkoxy-N-(3-Chloro-4-methoxyphenyl)-N′ cyclo heptyl-[1,3,5]triazine-2, 4 diamine

To6-chloro-N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine(5 g) dissolved in solvent (Methanol, Ethanol, Isopropanol) was addedK₂CO₃ (3.2 g) and refluxed for about 8-10 h. TLC was monitored. Aftercompletion of the reaction, the reaction mass was filtered throughvacuum and the filtrate was evaporated to yield the title compounds,E32, E33, and E34, respectively.

Rxn No. Solvent Yield (%) Purity (%) 1 Methanol 90 95 2 Ethanol 85 96 3Isopropanol 50 80

Example 132

Biological Activity Data

Additional bioactivity data for compounds of the present invention arepresented in Table 8, where compounds that have at least the activity ofeffecting cellular proliferation or at least the activity of modulatinginflammation activity, as measured by the assays taught herein, areprovided. For compounds that show at least the activity of modulatinginflammation activity, the activity is typically measured and reportedrelative to IL6 production compared to cells that did not receivecompound (or percent of control IL6 production). The inclusion ofcompounds in the categories of the Tables disclosed herein, includingthis Table, is not to be seen as limiting, in that compounds included insuch Tables have at least the activity shown for inclusion in the Tableand may have more or other activities. Nor are the Tables to be seen aslimiting in that these are the only compounds disclosed herein that havethat activity, representative compounds are shown in the Tables thathave at least that particular activity for inclusion in the Table. Oneor more compounds disclosed herein have at least an activity that hasutility in treatment of disease states. Note that any hydrogen atomsthat are required for any atom to attain its usual valence in astructure presented in Table 8, whether a carbon atom or a heteroatom,should be inferred if it is not specifically indicated.

REFERENCES

-   1. Brownlee et al., 101 ANN. INTERN. MED. 527-37 (1984)-   2. Yang et al., 91 PROC NATL. ACAD. SCI. USA 9436-40 (1994)-   3. Tanji et al., 11 J. AM. SOC. NEPHROL. 1656-66 (2000)-   4. Schmidt et al., 84 CIRC. RES. 489-97 (1999)-   5. Yamamoto et al., 902 ANN. N.Y. ACAD. SCI. 163-70 (2000)-   6. Yano et al., 8 DIABETES RES. CLIN. PRACT. 215-19 (1990)-   7. Cohen et al., Vol. 7 J. AM. SOC. NEPHROL. 183-90 (1996)-   8. Brownlee et al., 232 SCIENCE 1629-32 (1986)-   9. Li et al., 93 PROC. NATL. ACAD. SCI. USA 3902-07 (1996)-   10. Piercy et al. 47 METABOLISM 1477-80 (1998)-   11. Wautier et al., 97 J. CLIN. INVEST. 238-43 (1996)-   12. Schmidt et al., 96 J. CLIN. INVEST. 1395-1403 (1995)-   13. Park et al., 4 NAT. MED. 1025-31 (1998)-   14. Taguchi et al., 405 NATURE 354-60 (2000)-   15. Hofmann et al., 97 CELL 889-901 (1999)-   16. Du Yan et al., 94 PROC. NATL. ACAD. SCI. U.S.A. 5296-5301 (1997)-   17. Lander et al., 272 J. BIOL. CHEM. 7810-4 (1997).-   18. Thornalley, 44 CELL MOL. BIOL. 1013-23 (1998)-   19. Orford et al., 86 AM. J. CARDIOL. 6H-11H. (2000)-   20. Bult, 21 PHARMACOL. SCI. 274-79 (2000)-   21. Schwartz, 100 J. CLIN. INVEST. S87-89 (1997)-   22. Cooper et al., 43 DIABETOLOGIA 660-64 (2000)-   23. Birrell et al., 4 DIABETOLOGIA 110-16 (2000)-   24. Wada et al., 42 DIABETOLOGIA 743-47 (1999)-   25. Soulis et al., 50 KIDNEY INT. 627-34 (1996)-   26. Cooper et al., 43 DIABETOLOGIA 660-64 (2000)-   27. Oturai et al., 49(8) METABOLISM 996-1000 (2000)-   28. Laight et al., 15 DIABETES METAB. RES. REV. 274-82 (1999)-   29. Stehouwer et al., 34 CARDIOVASC. 55-68 (1997)-   30. Libby, 247 J. INTERN. MED. 349-58 (2000)-   31. Van Lente, 293 CLINICA. CHIMICA. ACTA. 31-52 (2000)-   32. Horii et al., 39 KIDNEY INT. SUPPL. S71-75 (1993)-   33. Huber et al., 19 ARTERIOSCLERTHROMB. VASC. BIOL. 2364-67 (1999)-   34. Shikano et al., 85 NEPHRON 81-5 (2000)-   35. Pickup et al., 8(67) LIFE SCI. 291-300 (2000)-   36. Kado et al., 36 ACTA. DIABETOL. 67-72 (1999)-   37. Eitner et al., 51 KIDNEY INT. 69-78 (1997)-   38. Banba et al. 58 KIDNEY INT. 684-690 (2000)-   39. Campbell et al., 48 ANNU. REV. PHYSIOL. 295-306 (1986)-   40. Kamovsky, 105 ROUS—WHIPPLE AWARD LECTURE. AM. J. PATHOL. 200-206    (1981)-   41. Gustafson et al., 54 TETRAHEDRON 4051-4065 (1998)-   42. Shah et al., 2 J. COMB. CHEM. 453-460 (2000)-   43. Johnson et al., 54 TETRAHEDRON 4097-4106 (1998)-   44. Ichihara et al., CHEM. LETT. 631-632 (1995)-   45. Kaiser et al., 73 J. AM. CHEM. SOC. 2984-2986 (1951)-   46. Thurston et al., 73 J. AM. CHEM. SOC. 2981-2983 (1951)-   47. Campbell et al., 26 ORG. CHEM. 2786-2789 (1961)-   48. Koopman et al., 77 RECUEIL 235-240 (1958)-   49. Whitten et al., 39 J. MED. CHEM. 4354-4357 (1996)-   50. JP Patent No. 2001-145355 (issued Jan. 12, 2201)-   51. Masquelin et al., 48 HETEROCYCLES, 2489-2505 (1998)-   52. Scham et al., 2 J. COMB. CHEM. 361-369 (2000)-   53. Silen et al., 42 ANTIMICROB. AGENTS CHEMOTHER. 1447-53 (1998)-   54. Scham et al., 66 J. ORG. CHEM. 507-513 (2001)-   55. Masala et al., 1 ORG. LETT. 1355-1357 (1999)-   56. Miyaura et al., 95 CHEM. REV. 2457-2483 (1995)-   57. Suzuki et al., 576 ORGANOMET. CHEM. 147-168 (1999)-   58. Antilla et al., 3 ORG. LETT. 2077-2079 (2001)-   59. Janietz et al., SYNTHESIS, 33-34 (1993)

TABLE 1A Representative Compounds of the Present Invention CmpdStructure Name A1

N-Cycloheptyl-N′-(3-iodo-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA2

N-Cycloheptyl-N′-(4-methoxy-3-nitro-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA3

5-{4-Cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-methoxy-benzonitrileA4

N-Cycloheptyl-N′-(4-ethoxy-3-fluoro-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA5

N-Cycloheptyl-N′-(3-fluoro-4-propoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA6

N-Cycloheptyl-N′-(3-fluoro-4-isopropoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA7

N-(3-Chloro-4-ethoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA8

N-(3-Chloro-4-propoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA9

N-(3-Chloro-4-isopropoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA10

N-Cycloheptyl-N′-(4-methoxy-3-trifluoromethyl-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA11

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-benzyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA12

N-(3-Chloro-4-methoxy-benzyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA13

N-Cycloheptyl-N′-[2-(3-fluoro-4-methoxy-phenyl)-ethyl]-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA14

N-[2-(3-Chloro-4-methoxy-phenyl)-ethyl]-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA15

N-Cycloheptyl-N′-(3-fluoro-4-trifluoromethyl-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA16

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA17

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA18

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-benzyl)-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA19

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-benzyl)-N″-methyl-N″-piperidin-4-yl-[1,3,5]triazine-2,4,6-triamineA20

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N′,N″-dimethyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA21

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-methyl-N″-(4-methyl-cyclohexyl)-[1,3,5]triazine-2,4,6-triamineA22

N-Cycloheptyl-N′-cyclohexyl-N″-(3-fluoro-4-methoxy-phenyl)-N′,N″-dimethyl-[1,3,5]triazine-2,4,6-triamineA23

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N′,N″-dimethyl-N″-(1-methyl-piperidin-3-yl)-[1,3,5]triazine-2,4,6-triamineA24

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N′,N″-dimethyl-N″-piperidin-3-yl-[1,3,5]triazine-2,4,6-triamineA25

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N′,N″-dimethyl-N″-(1-methyl-1,4-dihydro-pyridin-4-yl)-[1,3,5]triazine-2,4,6-triamineA26

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N′,N″-dimethyl-N″-pyridin-4-yl-[1,3,5]triazine-2,4,6-triamineA27

4-({4-Cycloheptylamino-6-[(3-fluoro-4-methoxy-phenyl)-methyl-amino]-[1,3,5]triazin-2-yl}-methyl-amino)-1-methyl-pyridinium;chloride A28

Acetic acid4-{4-cyclo-heptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-fluoro-phenylesterA29

N-Cycloheptyl-N′-(5-fluoro-6-methoxy-pyridin-3-yl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA30

N-Cycloheptyl-N′-(4-fluoro-5-methoxy-pyridin-2-yl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA31

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-methyl-N″-(4-methyl-piperazin-1-yl)-[1,3,5]triazine-2,4,6-triamineA32

N-Cycloheptyl-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-(4-trifluoromethoxy-3-trifluoromethyl-phenyl)-[1,3,5]triazine-2,4,6-triamineA33

N-(4-Amino-3-fluoro-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA34

N-Cycloheptyl-N′-(3-fluoro-4-methylamino-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA35

N-Cycloheptyl-N′-(4-dimethylamino-3-fluoro-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA36

6-(3-Chloro-4-methyl-amino-phenoxy)-N-cycloheptyl-N′-methyl-N′-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4-diamineA37

N-(3-Chloro-4-dimethyl-amino-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA38

N-Cycloheptyl-6-(3-fluoro-4-methoxy-phenoxy)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-[1,3,5]-triazine-2,4-diamineA39

6-(3-Chloro-4-methoxy-phenoxy)-N-cycloheptyl-N′-methyl-N′-(1-methyl-piperidin-4-yl)-[1,3,5]-triazine-2,4-diamineA40

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N,N″-dimethyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA41

N-Cyclohept-4-enyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA42

N-Cycloheptyl-N′-(3-fluoro-4-methyl-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA43

N-Cycloheptyl-N′-(4-ethyl-3-fluoro-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA44

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-cyclohexyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA45

N-Cyclohept-3-enyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA46

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N,N″-dimethyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA47

4-{4-Cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-fluoro-phenolA48

Sodium;4-{4-(1-ethyl-hexylamino)-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-fluoro-phenolateA49

N-Cyclohept-2-enyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA50

2-Chloro-4-[4-cycloheptyl-amino-6-(1-methyl-piperidin-4-ylamino)-[1,3,5]triazin-2-ylamino]-phenolA51

4-[4-Cycloheptylamino-6-(1-methyl-piperidin-4-ylamino)-[1,3,5]triazin-2-ylamino]-2-fluoro-phenolA52

N-(3-Chloro-4-methoxy-phenyl)-N′-cyclohept-2-enyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA53

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-methyl-N″-piperidin-4-yl-[1,3,5]triazine-2,4,6-triamineA54

N-(3-Bromo-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA55

4-[4-Cycloheptylamino-6-(methyl-piperidin-4-yl-amino)-[1,3,5]triazin-2-ylamino]-2-fluoro-phenolA56

2-Chloro-4-[4-cycloheptyl-amino-6-(methyl-piperidin-4-yl-amino)-[1,3,5]triazin-2-ylamino]-phenolA57

Sodium;2-chloro-4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-phenolateA58

N-(3-Chloro-4-methoxy-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA59

Phosphoric acidmono-(2-chloro-4-{4-cycloheptyl-amino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-phenoxymethyl)ester A60

Monosodium salt,phosphoric acidmono-(2-chloro-4-{4-cycloheptyl-amino-6-[methyl-(1-methyl-piperidin-4-yl)-amino-[1,3,5]triazine-2-ylamino}-phenoxymethyl)ester A61

N-(3-Chloro-4-methyl-sulfanylmethoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA62

Phosphoric acid di-tert-butyl ester2-chloro-4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-phenoxymethylester A63

Sodium;2-chloro-4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-phenolateA64

N-Cycloheptyl-N′-(3-fluoro-4-methoxymethoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA65

Phosphoric acid dibenzylester2-chloro-4-{4-cyclo-heptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-phenoxymethylesterA66

Phosphoric acidmono-(4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-fluoro-phenoxymethyl)esterA67

2-(2-{2-[2-(2-Chloro-4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-phenoxy)-ethoxy]-ethoxy}-ethoxy)-ethanolA68

2-(2-{2-[2-(4-{4-Cyclo-heptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-fluoro-phenoxy)-ethoxy]-ethoxy}-ethoxy)-ethanolA69

4-{[4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazin-2-yl]-methyl-amino}-1-methyl-1-phosphonooxymethyl-piperidinium;chloride A70

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-methyl-N″-pyridin-4-yl-[1,3,5]triazine-2,4,6-triamineA71

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-pyridin-4-yl-[1,3,5]triazine-2,4,6-triamineA72

Phosphoric acidmono-(4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-fluoro-phenyl)ester A73

Phosphoric acidmono-(4-{[4-cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-yl]-methyl-amino}-piperidin-1-ylmethyl)ester A74

4-{[4-Cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-yl]-methyl-amino}-1-methyl-1-phosphonooxymethyl-piperidinium;chloride A75

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-pyridin-4-yl-[1,3,5]triazine-2,4,6-triamineA76

4-{[4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazin-2-yl]-methyl-amino}-1-methyl-pyridinium;chloride A77

4-{[4-Cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-yl]-methyl-amino}-1,1-dimethyl-piperidinium;iodide A78

N-Allyl-N′-(3-chloro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA79

N-(3-Chloro-4-methoxy-phenyl)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-propyl-[1,3,5]triazine-2,4,6-triamineA80

N-(3-Fluoro-4-methoxy-phenyl)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-propyl-[1,3,5]triazine-2,4,6-triamineA81

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(3-methylamino-propyl)-[1,3,5]triazine-2,4,6-triamineA82

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-(3-dimethylamino-propyl)-N″-methyl-[1,3,5]triazine-2,4,6-triamineA83

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-(3-dimethylamino-propyl)-[1,3,5]triazine-2,4,6-triamineA84

N-Cycloheptyl-N′-(3-dimethylamino-propyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamineA85

6-Azepan-1-yl-N-(3-chloro-4-methoxy-phenyl)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4-diamineA86

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4,6-triamineA87

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-methoxymethyl-[1,3,5]triazine-2,4,6-triamineA88

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-piperidin-1-yl-[1,3,5]triazine-2,4,6-triamineA89

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-(1-ethyl-piperidin-4-yl)-N″-methyl-[1,3,5]triazine-2,4,6-triamineA90

4-{[4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazin-2-yl]-methyl-amino}-piperidine-1-carbaldehydeA91

4-{[4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazin-2-yl]-methyl-amino}-cyclohexanolA92

N-(1-Aza-bicyclo[2.2.2]oct-3-yl)-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-triamineA93

N-Bicyclo[2.2.2]oct-2-yl-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-triamineA94

N-(1-Aza-bicyclo[2.2.2]oct-3-yl)-N′-cycloheptyl-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamineA95

N-Bicyclo[2.2.2]oct-2-yl-N′-cycloheptyl-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamineA96

N-Bicyclo[2.2.1]hept-2-yl-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-triamineA97

N-(3-Chloro-4-methoxy-phenyl)-N′-cyclo-heptylidene-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA98

N-(4-Amino-cyclohexyl)-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-N-methyl-[1,3,5]triazine-2,4,6-triamineA99

N-(4-Amino-cyclohexyl)-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-triamineA100

3-{[4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazin-2-yl]-methyl-amino}-cyclohexanolA101

3-{[4-Cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-yl]-methyl-amino}-cyclohexanolA102

N-(3-Amino-cyclohexyl)-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-N-methyl-[1,3,5]triazine-2,4,6-triamineA103

N-(4-Amino-cyclohexyl)-N′-cycloheptyl-N″-(3-fluoro-4-methoxy-phenyl)-N-methyl-[1,3,5]triazine-2,4,6-triamineA104

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-(4-dimethylamino-cyclohexyl)-N″-methyl-[1,3,5]triazine-2,4,6-triamineA105

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-(4-methoxy-cyclohexyl)-N″-methyl-[1,3,5]triazine-2,4,6-triamineA106

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-(4-methoxy-cyclohexyl)-N″-methyl-[1,3,5]triazine-2,4,6-triamineA107

ohexyl)-[1,3,5]triazine-2,4,6-triamine A108

4-[4-(3-Chloro-4-methoxy-phenylamino)-6-cyclo-heptylamino-[1,3,5]triazin-2-ylamino]-cyclohexanethiolA109

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-(4-methylsulfanyl-cyclohexyl)-[1,3,5]triazine-2,4,6-triamineA110

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-(4-methanesulfonyl-cyclo-hexyl)-[1,3,5]triazine-2,4,6-triamineA111

clohexyl)-[1,3,5]triazine-2,4,6-triamine A112

N-(3-Chloro-4-methoxy-phenyl)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-thiepan-4-yl-[1,3,5]triazine-2,4,6-triamineA113

6-(Bicyclo[2.2.1]hept-2-yloxy)-N-(3-chloro-4-methoxy-phenyl)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4-diamineA114

N-(3-Chloro-4-methoxy-phenyl)-N′-(1,1-dioxo-1lambda*6*-thiepan-4-yl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA115

N-(1,1-Dioxo-1lambda*6*-thiepan-4-yl)-N′-(3-fluoro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA116

N-(3-Chloro-4-methoxy-phenyl)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-thiepan-3-yl-[1,3,5]triazine-2,4,6-triamineA117

5{[4-Cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-methyl}-thiazolidine-2,4-dioneA118

5-{[4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazin-2-ylamino]-methyl}-thiazolidine-2,4-dioneA119

5-{[4-Cycloheptylamino-6-(3-fluoro-4-hydroxy-phenylamino)-[1,3,5]triazin-2-ylamino]-methyl}-thiazolidine-2,4-dioneA120

5-{[4-(3-Chloro-4-hydroxy-phenylamino)-6-cyclo-heptylamino-[1,3,5]triazin-2-ylamino]-methyl}-thiazolidine-2,4-dioneA121

6-(Bicyclo[2.2.1]hept-2-yloxy)-N-(3-fluoro-4-methoxy-phenyl)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4-diamineA122

4-[4-Cycloheptylamino-6-(hydroxymethyl-amino)-[1,3,5]triazin-2-ylamino]-2-fluoro-phenolA123

5-({4-(3-Fluoro-4-methoxy-phenylamino)-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-methyl)-thiazolidine-2,4-dioneA124

Sodium;4-[4-cycloheptyl-amino-6-(pyrrolidine-2-carbonyloxy)-[1,3,5]triazin-2-ylamino]-2-fluoro-phenolateA125

N-Cycloheptyl-N′-(2,3-dihydro-1H-indol-2-ylmethyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamineA126

4-[4-Cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-cyclo-hexanethiolA127

2-{[4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazin-2-yl]-methyl-amino}-cyclohexanolA128

N-(2-Amino-cyclohexyl)-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-N-methyl-[1,3,5]triazine-2,4,6-triamineA129

N-(2-Amino-cyclohexyl)-N′-cycloheptyl-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamineA130

4-[4-(2-Amino-cyclohexyl-amino)-6-cycloheptyl-amino-[1,3,5]triazin-2-ylamino]-2-fluoro-phenolA131

2-[4-Cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-cyclohexanolA132

N-(3-Fluoro-4-methoxy-phenyl)-N′-(2-methyl-cyclohexyl)-N″-(1-methyl-hexyl)-[1,3,5]triazine-2,4,6-triamineA133

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-(3-methyl-cyclohexyl)-[1,3,5]triazine-2,4,6-triamineA134

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-(3-methyl-cyclohexyl)-[1,3,5]triazine-2,4,6-triamineA135

3-(2-{2-[2-(2-Hydroxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionicacid4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-fluoro-phenylester A136

3-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionicacid4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-fluoro-phenylester A137

3-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid2-chloro-4-{4-cycloheptyl-amino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-phenylester A138

3-(2-{2-[2-(2-Hydroxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid2-chloro-4-{4-cycloheptyl-amino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-phenylester A139

1-{4-[4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazin-2-ylamino]-piperidin-1-yl}-3-(2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propan-1-oneA140

1-{4-[4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazin-2-ylamino]-piperidin-1-yl}-3-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propan-1-oneA141

1-{4-[4-Cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-piperidin-1-yl}-3-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propan-1-oneA142

1-{4-[4-Cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-piperidin-1-yl}-3-(2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propan-1-oneA143

4-[4-Cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-1-{2-[2-(2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethyl}-1-methyl-piperidinium;chlorideA144

4-[4-(3-Chloro-4-methoxy-phenylamino)-6-cyclo-heptylamino-[1,3,5]triazin-2-ylamino]-1-{2-[2-(2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethyl}-1-methyl-piperidinium;chloride A145

4-[4-(3-Chloro-4-methoxy-phenylamino)-6-cyclo-heptylamino-[1,3,5]triazin-2-ylamino]-1-{2-[2-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethyl}-1-methyl-piperidinium;chloride A146

4-[4-Cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-1-{2-[2-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethyl}-1-methyl-piperidinium;chlorideA147

5-{4-Cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-methoxy-benzoicacid A148

5-{4-Cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-methoxy-benzoicacidethyl ester A149

Sodium;5-{4-cyclo-heptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-methoxy-benzoateA150

4-{[4-Cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-yl]-methyl-amino}-1-methyl-piperidinium;chloride A151

4-{[4-Cycloheptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-yl]-methyl-amino}-1-methyl-piperidinium;bromide A152

4-{[4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazin-2-yl]-methyl-amino}-1-methyl-piperidinium;bromide A153

3-Carboxy-propionate4-{[4-(3-chloro-4-methoxy-phenylamino)-6-cyclo-heptylamino-[1,3,5]triazin-2-yl]-methyl-amino}-1-methyl-piperidinium;A154

3-Carboxy-acrylate4-{[4-(3-chloro-4-methoxy-phenylamino)-6-cyclo-heptylamino-[1,3,5]triazin-2-yl]-methyl-amino}-1-methyl-piperidinium;A155

Carboxy-methane-carboxylate4-{[4-(3-chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazin-2-yl]-methyl-amino}-1-methyl-piperidinium;A156

Carboxy-methane-carboxylate4-{[4-cyclo-heptylamino-6-(3-fluoro-4-methoxy-phenylamino)-[1,3,5]triazin-2-yl]-methyl-amino}-1-methyl-piperidinium;A157

3-Carboxy-acrylate4-{[4-(3-chloro-4-methoxy-phenylamino)-6-cyclo-heptylamino-[1,3,5]triazin-2-yl]-methyl-amino}-1-methyl-piperidinium;A158

4-[4-(3-Chloro-4-hydroxy-phenylamino)-6-cyclo-heptylamino-[1,3,5]triazin-2-ylamino]-piperidinium;chlorideA159

4-[4-Cycloheptylamino-6-(3-fluoro-4-hydroxy-phenylamino)-[1,3,5]triazin-2-ylamino]-piperidinium;chloride A160

4-{4-Cycloheptylamino-6-[1-(4-methoxy-benzyl)-piperidin-4-ylamino]-[1,3,5]triazin-2-ylamino}-2-fluoro-phenolA161

4-[4-Cycloheptylamino-6-(piperidin-4-ylamino)-[1,3,5]triazin-2-ylamino]-2-fluoro-phenolA162

4-[4-Cyclopropylamino-6-(piperidin-4-ylamino)-[1,3,5]triazin-2-ylamino]-2-fluoro-phenolA163

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-1-oxy-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA164

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methyl-1-oxy-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA165

4-{4-Cycloheptylamino-6-[methyl-(1-methyl-1-oxy-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-2-fluoro-phenolA166

2-Chloro-4-{4-cyclo-heptylamino-6-[methyl-(1-methyl-1-oxy-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-phenolA167

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-N″-(1-methanesulfonyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA168

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-(1-methanesulfonyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamineA169

A170

A171

A172

A173

A174

A175

A176

A177

A178

TABLE 2 Representative compounds of the present invention prepared byparallel synthesis reactions, including amine monomers, products, andcharacterization data Cmpd No. R1 (Monomer 1) R2 (Monomer 2) R3 (Monomer3) Product (Structure) 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

LC-MS (Calc′d MW) Synthetic Observed MW Yield Based Purification CmpdNo. Product Name Procedure (M + H) LC Purity on Purity method 1N2-(4-bromo-1-naphthyl)-N4- Parallel synthesis (538) 540.4 62 23 ISCOcycloheptyl-N6-[(1-ethyl-2- method Apyrrolidinyl)methyl]-1,3,5-triazine- 2,4,6-triamine 2N2-(4-chloro-1-naphthyl)-N4- Parallel synthesis (493) 494.3 54 18 ISCOcycloheptyl-N6-[(1-ethyl-2- method Apyrrolidinyl)methyl]-1,3,5-triazine- 2,4,6-triamine 3N2-cycloheptyl-N4-[(1-ethyl-2- Parallel Synthesis (460) 461.5 95 25 ISCOpyrrolidinyl)methyl]-N6-(3- method A quinolinyl)-1,3,5-triazine-2,4,6-triamine 4 N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (460) 461.497 26 ISCO pyrrolidinyl)methyl]-N6-(6- method Aquinolinyl)-1,3,5-triazine-2,4,6- triamine 5N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (460) 461.4 98 61 ISCOpyrrolidinyl)methyl]-N6-(8- method A quinolinyl)-1,3,-triazine-2,4,6-triamine 6 N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (487) 488.594 39 ISCO pyrrolidinyl)methyl]-N6-[1-(2- method Anaphthyl)ethyl]-1,3,5-triazine- 2,4,6-triamine 7 N2-cycloheptyl-N4-(3,4-Parallel synthesis (478) 478.3 94 53 ISCOdichlorophenyl)-N6-[(1-ethyl-2- method Apyrrolidinyl)methyl]-1,3,5-triazine- 2,4,6-triamine 8N2-cycloheptyl-N4-(3,4- Parallel synthesis 445 (446.3) 98 55 ISCOdifluorophenyl)-N6-[(1-ethyl-2- method Apyrrolidinyl)methyl]-1,3,5-triazine 2,4,6-triamine 9N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (493) 494.3 96 40 ISCOpyrrolidinyl)methyl]-N6-[4- method A (triflouromethoxy)phenyl]-1,3,5-triazine-2,4,6-triamine 10 N2-cycloheptyl-N4-[(1-ethyl-2- Parallelsynthesis (493) 428.4 90 37 ISCO pyrrolidinyl)methyl]-N6-(4- method Afluorophenyl)-1,3,5-triazine-2,4,6- triamine 114-[(4-(cycloheptylamino)-6-([(1- Parallel synthesis (434) 435.4 76 64none ethyl-2-pyrrolidinyl)methyl]amino)- method A 1,3,5-triazin-2-yl)-amino]benzonitrile 12 N2-(4-chlorophenyl)-N4- Parallel synthesis (444)444.3 76 69 none cycloheptyl-N6-[(1-ethyl-2- method Apyrrolidinyl)methyl]-1,3,5-triazine- 2,4,8-triamine 13N2-(4-bromophenyl)-N4- Parallel synthesis (488) 488.4 77 70 nonecycloheptyl-N6-[(1-ethyl-2- method Apyrrolidinyl)methyl]-1,3,5-triazine- 2,4,6-triamine 14N4-[(4-(cycloheptylamino)-6-{[(1- Parallel synthesis (481) 482.5 96 46ISCO ethyl-2-pyrrolidinyl)methyl]amino}- method A1,3,5-triazin-2-yl)-amino]benzoate 15 N2-(1,1′-biphenyl-4-yl)-N4-Parallel synthesis (485) 486.5 75 69 none cycloheptyl-N6-[(1-ethyl-2-method A pyrrolidinyl)methyl]-1,3,5-triazine- 2,4,6-triamine 16N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (427) 428.3 78 75 nonepyrrolidinyl)methyl]-N6-(3- method A fluorophenyl)-1,3,5-triazine-2,4,6-triamine 17 N2-(3-chlorophenyl)-N4- Parallel synthesis (444) 444.3 77 74none cycloheptyl-N6-[(1-ethyl-2- method Apyrrolidinyl)methyl]-1,3,5-triazine- 2,4,6-triamine 18N2-(3-bromophenyl)-N4- Parallel synthesis (488) 488.4 82 75 nonecycloheptyl-N6-[(1-ethyl-2- method Apyrrolidinyl)methyl]-1,3,5-triazine- 2,4,6-triamine 193-[(4-(cycloheptylamino)-6-{[(1- Parallel synthesis (481) 482.4 98 45ISCO ethyl-2-pyrrolidinyl)methyl]amino}- method A1,3,5-triazin-2-yl)-amino]benzoate 20 N2-cycloheptyl-N4-[(1-ethyl-2-Parallel synthesis (427) 428.4 60 23 ISCO pyrrolidinyl)methyl]-N6-(2-method A fluorophenyl)-1,3,5-triazine-2,4,6- triamine 21N2-(2-chlorophenyl)-N4- Parallel synthesis (444) 444.3 53 26 ISCOcycloheptyl-N6-[(1-ethyl-2- method A pyrrolidinyl)methyl]-1,3,5-triazine2,4,6-triamine 22 N2-(2-bromophenyl)-N4- Parallel synthesis (488) 490.351 17 ISCO cycloheptyl-N6-[(1-ethyl-2- method Apyrrolidinyl)methyl]-1,3,5-triazine- 2,4,6-triamine 23N2-(1,3-benzodioxol-5-yl)-N4- Parallel synthesis (453) 454.4 16 4 ISCOcycloheptyl-N6-[(1-ethyl-2- method Apyrrolidinyl)methyl]-1,3,5-triazine- 2,4,6-triamine 24N2-cycloheptyl-N4-(2,3-dihydro- Parallel synthesis (467) 454.4 91 30ISCO 1,4-benzodioxin-6-yl)-N6-[(1-ethyl method A2-pyrrolidinyl)methyl]-1,3,5- triazine-2,4,6-triamine 25N2-cycloheptyl-N4-[4- Parallel synthesis (452) 453.5 67 17 ISCO(dimethylamino)phenyl]-N6-[(1- method Aethyl-2-pyrrolidinyl)methyl]-1,3,5- triazine-2,4,6-triamine 26N2-[3-chloro-4- Parallel synthesis (515) 515.5 91 45 ISCO(diethylamino)phenyl-N4- method A cycloheptyl-N6-[1-ethyl-2-pyrrolidinyl)methyl]1,3,5-triazine- 2,4,6-triamine 27N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (494) 495.4 92 41 ISCOpyrrolidinyl)methyl]-N6-[4-(4- method Amorpholinyl)phenyl]-1,3,5-triazine- 2,4,6-triamine 28N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (507) 508.5 4 0.004none pyrrolidinyl)methyl]-N6-[4-(4- method Amethyl-1-piperazinyl)phenyl]- 1,3,5-triazine-2,4,6-triamine 29N-[4-[(4-(cycloheptylamino)-6-{[(1- Parallel synthesis (466) 467.3 91 26ISCO ethyl-2-pyrrolidinyl)methy]amino}- method A 1,3,5-triazin-2-yl)-amino]phenyl)acetamide 30 N-{3-[(4-(cycloheptylamino)-6-{[(1- Parallelsynthesis (466) 467.4 100 38 ISCO ethyl-2-pyrrolidinyl)methyl]amino}-method A 1,3,5-triazin-2-yl)- amino]phenyl}acetamide 31N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (439) 440.4 96 42 ISCOpyrrolidinyl)methyl]-N6-(3- method A methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 32 N2-cycloheptyl-N4-(4- Parallel synthesis (453) 454.297 47 ISCO ethoxyphenyl)-N6-[(1-ethyl-2- method Apyrrolidinyl)methyl]-1,3,5-triazine- 2,4,6-triamine 33N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (455) 456.4 93 49 ISCOpyrrolidinyl)methyl]-N6-[4- method A (methylthio)phenyl]-1,3,5-triazine-2,4,6-triamine 34 N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis(410) 411.3 27 7 ISCO pyrrolidinyl)methyl]-N6-(2- method Apyridinyl)-1,3,5-triazine-2,4,6- triamine 35N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (423) 72 76 nonepyrrolidinyl)methyl]-N6-(2- method A methylphenyl)-1,3,5-triazine-2,4,6-triamine 36 N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (501)502.3 100 46 ISCO pyrrolidinyl)methyl]-N6-(4- method Aphenoxyphenyl)-1,3,5-triazine- 2,4,6-triamine 37N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (423) 424.4 86 46 ISCOpyrrolidinyl)methyl]-N6-(3- method A methylphenyl)-1,3,5-triazine-2,4,6-triamine 38 N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (423)424.3 98 44 ISCO pyrrolidinyl)methyl]-N6-(4- method Amethylphenyl)-1,3,5-triazine-2,4,6- triamine 392-[(4-(cycloheptylamino)-6-{[(1- Parallel synthesis (472) 473.3 63 16ISCO ethyl-2-pyrrolidinyl)methyl]amino)- method A1,3,5-triazin-2-yl)-amino]-4- methyl-3-thiophenecarboxamide 40N2-(4-chlorophenyl)-N4- Parallel synthesis (458) 458.5 98 42 ISCOcycloheptyl-N6-[(1-ethyl-2- method A pyrrolidinyl)methyl]-N2-methyl-1,3,5-triazine-2,4,6-triamine 41 3-[(4-(cycloheptylamino)-6-{[(1-Parallel synthesis (462) 463.4 97 45 ISCOethyl-2-pyrrolidinyl)methyl]amino}- method A 1,3,5-triazin-2-yl)-(phenyl)amino]propanenitrile 42 N2-cycloheptyl-N4-[(1-ethyl-2- Parallelsynthesis (543) 454.4 89 36 ISCO pyrrolidinyl)methyl]-N6-(4- method Amethoxyphenyl)-N6-methyl-1,3,5- triazine-2,4,6-triamine 43N2-cycloheptyl-N4-(2,4- Parallel synthesis (459) 460.3 73 74 nonedifluorophenyl)-N6-[(1-ethyl-2- method A pyrrolidinyl)methyl]-N4-methyl-1,3,5-triazine-2,4,6-triamine 44 [(4-(cycloheptylamino)-6-{[(1- Parallelsynthesis (448) 449.3 85 76 none ethyl-2-pyrrolidinyl)methyl]amino}-method A 1,3,5-triazin-2- yl)(phenyl)amino]acetonitrile 45N2-(3-chlorophenyl)-N4- Parallel synthesis (458) 458.5 98 37 ISCOcycloheptyl-N6-[(1-ethyl-2- method A pyrrolidinyl)methyl]-N2-methyl-1,3,5-triazine-2,4,6-triamine 46 N2-cycloheptyl-N4-[(1-ethyl-2- Parallelsynthesis (491) 492.3 82 21 ISCO pyrrolidinyl)methyl]-N6-methyl-N6-method A [2-(trifluoromethyl)phenyl]-1,3,5- triazine-2,4,6-triamine 47N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (507) 508.3 100 41ISCO pyrrolidinyl)methyl]-N6-methyl-N6- method A[2-(trifluoromethoxy)phenyl]-1,3,5- triazine-2,4-6-triamine 48N2-(3-chloro-4-methoxyphenyl)- Parallel synthesis (474) 474.3 93 51 ISCON4-cycloheptyl-N6-[(1-ethyl-2- method Apyrrolidinyl)methyl]-1,3,5-triazine- 2,4,6-triamine 49 N-benzoyl-4-[(4-Parallel synthesis (592) 593.4 14 2 none(cycloheptylamino)-6-{[(1-ethyl-2- method Apyrrolidinyl)methyl]amino}-1,3,5- triazin-2-yl)-amino]benzenesulfonamide 50 N2-cycloheptyl-N4-[(1-ethyl-2- Parallelsynthesis (459) 460.4 83 36 ISCO pyrrolidinyl)methyl]-N6-(2- method Anaphthyl)-1,3,5-triazine-2,4,6- triamine 51 N²-ethyl-N⁴-[(1-ethyl-2-Parallel synthesis (389) 390.2 48 38 nonepyrrolidinyl)methyl]-N⁶-(3-fluoro-4- method Bmethoxyphenyl)-1,3,5-triazine- 2,4,6-triamine 52N²-(tert-butyl)-N⁴-[(1-ethyl-2- Parallel synthesis (417) 418.4 38 28none pyrrolidinyl)methyl]-N⁶-(3-fluoro-4- method Bmethoxyphenyl)-1,3,5-triazine- 2,4,6-triamine 53N²-benzyl-N⁴-[(1-ethyl-2- Parallel synthesis (451) 452.3 87 75 nonepyrrolidinyl)methyl]-N⁶-(3-fluoro-4- method Bmethoxyphenyl)-1,3,5-triazine- 2,4,6-triamine 54N²-cyclooctyl-N⁴-[(1-ethyl-2- Parallel synthesis (471) 472.4 83 66 nonepyrrolidinyl)methyl]-N⁶-(3-fluoro-4- method Bmethoxyphenyl)-1,3,5-triazine- 2,4,6-triamine 55N²-cyclohexyl-N⁴-[(1-ethyl-2- Parallel synthesis (443) 444.3 75 80 nonepyrrolidinyl)methyl]-N⁶-(3-fluoro-4- method Bmethoxyphenyl)-1,3,5-triazine- 2,4,6-triamine 56N²-cyclopentyl-N⁴-[(1-ethyl-2- Parallel synthesis (429) 430.3 79 75 nonepyrrolidinyl)methyl]-N⁶-(3-fluoro-4- method Bmethoxyphenyl)-1,3,5-triazine- 2,4,6-triamine 57N²-[(1-ethyl-2-pyrrolidinyl)methyl]- Parallel synthesis (415) 416.3 8445 ISCO N⁴-(3-fluoro-4-methoxyphenyl)-6- method B(1-pyrrolidinyl)-1,3,5-triazine-2,4- diamine 58N²-[(1-ethyl-2-pyrrolidinyl)methyl]- Parallel synthesis (443) 444.4 9051 ISCO N⁴-(3-fluoro-4-methoxyphenyl)-6- method Bhexahydro-1H-azepin-1-yl-1,3,5- triazine-2,4-diamine 59N-[(1-ethyl-2-pyrrolidinyl)methyl]- Parallel synthesis (483) 484.5 86 47ISCO N⁴-(3-fluoro-4-methoxyphenyl)-6- method Boctahydro-1(2H)-quinolinyl-1,3,5- triazine-2,4-diamine 60N²-[(1-ethyl-2-pyrrolidinyl)methyl]- Parallel synthesis 457 (458.4) 8850 ISCO N⁴-(3-fluoro-4-methoxyphenyl)- method BN⁶-(4-methylcyclohexyl)-1,3,5- triazine-2,4,6-triamine 61N²-(1-ethyl-pyrrolidin-2-ylmethyl]- Parallel synthesis (459) 460.4 81 36ISCO N⁴-(3-fluoro-4-methoxyphenyl)-6- method B((S)-2-methoxymethyl-pyrrolidin-1 yl)-1,3,5-triazine-2,4-diamine 62N²-[(1-ethyl-2-pyrrolidinyl)methyl]- Parallel synthesis (444) 445.3 3010 none N⁴-(3-fluoro-4-methoxyphenyl)-6- method B(4-methyl-1-piperazinyl)-1,3,5- triazine-2,4-diamine 636-(4-acetyl-1-piperazinyl)-N²-[(1- Parallel synthesis 472 (473.3) 83 43ISCO ethyl-2-pyrrolidinyl)methyl]-N⁴-(3- method Bfluoro-4-methoxyphenyl)-1,3,5- triazine-2,4-diamine 64 Ethyl4-{4-{[(1-ethyl-2- Parallel synthesis (502) 503.2 75 74 nonepyrrolidinyl)methyl]amino}-N⁶-[(3- method Bfluoro-4-methoxyphenyl)-amino]- 1,3,5-triazin-2-yl}-1-piperazinecarboxylate 65 N²-(cyclohexylmethyl)-N⁴-[(1- Parallelsynthesis (457) 458.4 76 77 none ethyl-2-pyrrolidinyl)methyl]-N⁶-(3-method B fluoro-4-methoxyphenyl)-1,3,5- triazine-2,4,6-triamine 66N²-[(1-ethyl-2-pyrrolidinyl)methyl]- Parallel synthesis (441) 442.2 7770 none N⁴-(3-fluoro-4-methoxyphenyl)- method BN⁶-(2-furylmethyl)-1,3,5-triazine- 2,4,6-triamine 67N²-[(1-ethyl-2-pyrrolidinyl)methyl]- Parallel synthesis (443) 444.1 7769 none N⁴-(3-fluoro-4-methoxyphenyl)- method BN⁶-(2,2,2-trifluoroethyl)-1,3,5- triazine-2,4,6-triamine 68N²-[2-(dimethylamino)ethyl]-N⁴- Parallel synthesis (432) 433.5 6 2 none[(1-ethyl-2-pyrrolidinyl)methyl]-N⁶- method B(3-fluoro-4-methoxyphenyl)-1,3,5- triazine-2,4,6-triamine 69N²-[(1-ethyl-2-pyrrolidinyl)methyl]- Parallel synthesis (541) 542.4 7839 ISCO N⁴-(3-fluoro-4-methoxyphenyl)- method B N⁶-{4-[2-oxo-2-(1-pyrrolidinyl)ethyl]-1-piperazinyl)- 1,3,5-triazine-2,4-diamine 70N²,N⁴-bis[(1-ethyl-2- Parallel synthesis (472) 473.3 63 20 ISCOpyrrolidinyl)methyl]-N⁶-(3-fluoro-4- method Bmethoxyphenyl)-1,3,5-triazine- 2,4,6-triamine 71N²-[(1-ethyl-2-pyrrolidinyl)methyl]- Parallel synthesis (472) 473.3 6728 ISCO N⁴-(3-fluoro-4-methoxyphenyl)- method BN⁶-[2-(1-piperidinyl)ethyl]-1,3,5- triazine-2,4,6-triamine 72N⁶-[4-(1,3-benzodioxol-5- Parallel synthesis (564) 565.4 87 83 noneylmethyl)-1-piperazinyl]-N²-[(1- method Bethyl-2-pyrrolidinyl)methyl]-N⁴-(3- fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4-diamine 73 N²-[(1-ethyl-2-pyrrolidinyl)methyl]- Parallelsynthesis (507) 508.3 86 41 ISCO N⁴-(3-fluoro-4-methoxyphenyl)- method BN⁶-[4-(2-pyridinyl)-1-piperazinyl]- 1,3,5-triazine-2,4-diamine 741-[3-({4-{[(1-ethyl-2- Parallel synthesis (486) 487.5 85 31 ISCOpyrrolidinyl)methyl]amino}-6-[(3- method Bfluoro-4-methoxyphenyl)amino]- 1,3,5-triazin-2-yl)amino)propyl]-2-pyrrolidinone 75 N²-[(1-ethyl-2-pyrrolidinyl)methyl]- Parallel synthesis(469) 470.3 90 38 ISCO N⁴-(3-fluoro-4-methoxyphenyl)- method BN⁶-[3-(1H-imidazol-1-yl)propyl]- 1,3,5-triazine-2,4,6-triamine 76N2-cycloheptyl-N4-ethyl-N6-(3- Parallel synthesis (374) 375.3 85 18preparative HPLC fluoro-4-methoxyphenyl)-1,3,5- method Ctriazine-2,4,6-triamine 77 N2-(tert-butyl)-N4-cycloheptyl-N6- Parallelsynthesis (402) 403.4 81 3 preparative HPLC(3-fluoro-4-methoxyphenyl)-1,3,5- method C triazine-2,4,6-triamine 78N2-benzyl-N4-cycloheptyl-N6-(3- Parallel synthesis (436) 436.9 96 15preparative HPLC fluoro-4-methoxyphenyl)-1,3,5- method Ctriazine-2,4,6-triamine 79 N2-cycloheptyl-N4-cyclooctyl-N6- Parallelsynthesis (456) 457.3 96 5 preparative HPLC(3-fluoro-4-methoxyphenyl)-1,3,5- method C triazine-2,4,6-triamine 80N2-cycloheptyl-N4-cyclohexyl-N6- Parallel synthesis (428) 429.3 100 10preparative HPLC (3-fluoro-4-methoxyphenyl)-1,3,5- method Ctriazine-2,4,6-triamine 81 N2-cycloheptyl-N4-cyclopentyl- Parallelsynthesis (414) 415.2 89 26 preparative HPLCN6-(3-fluoro-4-methoxyphenyl)- method C 1,3,5-triazine-2,4,6-triamine 82N2-cycloheptyl-N4-(3-fluoro-4- Parallel synthesis (400) 400.9 98 22preparative HPLC methoxyphenyl)-6-(1-pyrrolidinyl)- method C1,3,5-triazine-2,4-diamine 83 N2-cycloheptyl-N4-(3-fluoro-4- Parallelsynthesis (428) 429 96 5 preparative HPLC methoxyphenyl)-6-hexahydro-1H-method C azepin-1-yl-1,3,5-triazine-2,4- diamine 84N2-cycloheptyl-N4-(3-fluoro-4- Parallel synthesis (468) 469.3 67 21preparative HPLC methoxyphenyl)-6-octahydro- method C1(2H)-quinolinyl-1,3,5-triazine-2,4- diamine 85N2-cycloheptyl-N4-(3-fluoro-4- Parallel synthesis (442) 443.3 64 4preparative HPLC methoxyphenyl)-N6-(4- method Cmethylcyclohexyl)-1,3,5-triazine- 2,4,6-triamine 86N2-cycloheptyl-N4-(3-fluoro-4- Parallel synthesis (444) 444.9 100 17preparative HPLC methoxyphenyl)-6-[(2S)-2- method C(methoxymethyl)-1-pyrrolidinyl]- 1,3,5-triazine-2,4-diamine 87N2-cycloheptyl-N4-(3-fluoro-4- Parallel synthesis (429) 430.3 94 13preparative HPLC methoxyphenyl)-6-(4-methyl-1- method Cpiperazinyl)-1,3,5-triazine-2,4- diamine 886-(4-acetyl-1-piperazinyl)-N2- Parallel synthesis (457) Wrong MW 100compound not preparative HPLC cycloheptyl-N4-(3-fluoro-4- method Cobservered observered methoxyphenyl)-1,3,5-triazine- after prep diamineHPLC 89 ethyl-4-{4-(cycloheptylamino)-6- Parallel synthesis (487) 488.397 4 preparative HPLC [(3-fluoro-4- method C methoxyphenyl)amino]-1,3,5-triazin-2-yl}-1- piperazinecarboxylate 90 N2-cycloheptyl-N4- Parallelsynthesis (442) 100 16 preparative HPLC(cyclohexylmethyl)-N6-(3-fluoro-4- method Cmethoxyphenyl)-1,3,5-triazine- 2,4,6-triamine 91N2-cycloheptyl-N4-(3-fluoro-4- Parallel synthesis (426) 426.9 59 31preparative HPLC methoxyphenyl)-N6-(2- method Cfuranylmethyl)-1,3,5-triazine-2,4,6 triamine 92N2-cycloheptyl-N4-(3-fluoro-4- Parallel synthesis (428) 352.1 93 1preparative HPLC methoxyphenyl)-N6-(2,2,2- method Ctrifluoroethyl)-1,3,5-triazine-2,4,6- triamine 93 N2-cycloheptyl-N4-[2-Parallel synthesis (417) 418.3 76 11 preparative HPLC(dimethylamino)ethyl]-N6-(3- method C fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine 94 N2-cycloheptyl-N4-(3-fluoro-4- Parallelsynthesis (526) 527.3 27 15 preparative HPLCmethoxyphenyl)-6-(4-[2-oxo-(1- method Cpyrrolidinyl)ethyl]-1-piperazinyl)- 1,3,5-triazine-2,4-diamine 95N2-cycloheptyl-N4-[(1-ethyl-2- Parallel synthesis (457) 458.4 80 24preparative HPLC pyrrolidinyl)methyl]-N6-(3-fluoro-4- method Cmethoxyphenyl)-1,3,5-triazine- 2,4,6-triamine 96N2-cycloheptyl-N4-(3-fluoro-4- Parallel synthesis (457) 458.4 86 21preparative HPLC methoxyphenyl)-N6-[2-(1- method Cpiperidinyl)ethyl]-1,3,5-triazine- 2,4,6-triamine 976-[4-(1,3-benzodioxol-5- Parallel synthesis (549) 493.2 94 21preparative HPLC ylmethyl)1-piperpazinyl]-N2- method Ccycloheptyl-N4-(3-fluoro-4- methoxyphenyl)-1,3,5-triazine-2,4- diamine98 N2-cycloheptyl-N4-(3-fluoro-4- Parallel synthesis (492) incorrect MW70 compound not preparative HPLC methoxyphenyl)-6-[4-(2-pyridinyl)-method C observed observered 1-piperazinyl]-1,3,5-triazine-2,4- afterprep triamine HPLC 99 1-[3-({4-(cycloheptylamino)-6-[(3- Parallelsynthesis (471) 472.3 96 29 preparative HPLCfluoro-4-methoxyphenyl)amino]- method C1,3,5-triazin-2-yl)amino)propyl]-2- pyrrolidinone 100N2-cycloheptyl-N4-(3-fluoro-4- Parallel synthesis (454) 455.3 94 24preparative HPLC methoxyphenyl)-N6-[3-(1H- method Cimidazol-1-yl)propyl]-1,3,5- triazine-2,4,6-triamine

TABLE 3 Triazine compounds active in anti-proliferation assay(perlecan), generally useful in treating cardiovascular disordersassociated with proliferative activity Activity Patent # STRUCTURE CASNAME (note 1) Avg IC-50 160

HClN2-cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-N6-methyl-N6-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triaminehydrogen chloride ++ 4-5 95

N2-cycloheptyl-N4-(1-ethyl-pyrrolidin-2-ylmethyl)-N6-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine +7.3 26

N2-(3-chloro-4-diethylamino-phenyl)-N4-cycloheptyl-N6-(1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine +7.73 139

N2-cycloheptyl-N4-((S)-1-ethyl-pyrrolidin-2-ylmethyl)-N6-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine++ 4-5 140

N2-cycloheptyl-N4-((R)-1-ethyl-pyrrolidin-2-ylmethyl)-N6-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine++ 4-5 163

HClN2-(3-chloro-4-diethylamino-phenyl)-N4-cycloheptyl-N6-(1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triaminehydrogen chloride + 8-10 123

N2-(3-bromo-methoxy-phenyl)-N4-cycloheptyl-N6-methyl-N6-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine +170

HClN2-(3-bromo-methoxy-phenyl)-N4-cycloheptyl-N6-methyl-N6-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triaminehydrogen chloride + 8-9 137

N2-(3-chloro-methoxy-phenyl)-N4-cycloheptyl-N6-methyl-N6-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine++ 4-5 103

N2-(3-chloro-4-methoxy-phenyl)-N4-cyclohexylmethyl-N6-methyl-N6-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine +~7 uM 105

N2-(3-chloro-4-methoxy-phenyl)-N4-methyl-N4-(1-methyl-piperidin-4-yl)-N6-(1-propyl-butyl)-1,3,5-triazine-2,4,6-triamine +~7 uM 148

N2-(1-aza-bicyclo[2.2.2]oct-3-yl)-N4-(3-chloro-4-methoxy-phenyl)-N6-(1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine++ less than 5 uM 146

N²-(3-chloro-4-methoxy-phenyl)-N⁴-cycloheptyl-N⁶-methyl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine+++ ~2.2 138

2-chloro-4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl-amino]-1,3,5-triazin-2-ylamino}-phenol++ 4.03 uM 135

N2-(3-chloro-4-methoxy-phenyl)-N4-cyclohepty-N6-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine++ 5.83 uM 169

N2-cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-N6-methyl-N6-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triaminesuccinate + 7.2 uM 167

N2-cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-N6-methyl-N6-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triaminemaleate + 6.8 uM 159

N²-Cycloheptyl-N⁴-(3-fluoro-4-methoxy-phenyl)-N⁶-methyl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine++ 5.7-6.9 uM 125

6-chloro-N-cyclohexylmethyl-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine +10 uM 127

6-Chloro-N-cycloheheptyl-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine +10 uM NOTE 1: The activity scale is as follows (inclusive): (1) “+++” =IC50 <3 uM; (2) “++” = IC50 is 3-7 uM; and (3) “+” = IC50 is >7 uM.

TABLE 4A Triazine compounds that are toxic or cause cell death for usein oncology applications Patent Name Number STRUCTURE Generated byAutonom TM Toxicity 93

N2-cycloheptyl-N4-[2-(dimethylamino)ethyl]-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triaminetoxic 3

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(3-quinolinyl)-1,3,5-triazine-2,4,6-triaminetoxic 5

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(8-quinolinyl)-1,3,5-triazine-2,4,6-triaminetoxic 6

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-[1-(2-naphthyl)ethyl]-1,3,5-triazine-2,4,6-triaminetoxic 7

N2-cycloheptyl-N4-(3,4-dichlorophenyl)-N6-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triaminetoxic 8

N2-cycloheptyl-N4-(3,4-difluorophenyl)N6-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triaminetoxic 9

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-[4-(triflouromethoxy)phenyl]-1,3,5-triazine-2,4,6-triaminetoxic 12

N2-(4-chlorophenyl)-N4-cycloheptyl-N6-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triaminetoxic 13

N2-(4-bromophenyl)-N4-cycloheptyl-N6-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triaminetoxic 14

4-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-amino]benzoatetoxic 16

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(3-fluorophenyl)-1,3,5-triazine-2,4,6-triaminetoxic 21

N2-(2-chlorophenyl)-N4-cycloheptyl-N6-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triaminetoxic 22

N2-(2-bromophenyl)-N4-cycloheptyl-N6-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triaminetoxic 36

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(4-phenoxyphenyl)-1,3,5-triazine-2,4,6-triaminetoxic 47

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-methyl-N6-[2-(trifluoromethoxy)phenyl]-1,3,5-triazine-2,4,6-triaminetoxic 54

N2-cyclooctyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(4-fluoro-3-methoxyphenyl)-1,3,5-triazine-2,4,6-triaminetoxic 59

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(4-fluoro-3-methoxyphenyl)-6-octahydro-1(2H)-quinolinyl-1,3,5-triazine-2,4-diaminetoxic 121

N,N′-Bis-(3-chloro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]]triazine-2,4,6-triamineToxic by toxicityassay at 0.75 uM.Starts blunting by1 uM and roundingby5 uM 111

N-Butyl-N′-(3-chloro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperin-4-yl)-N-propyl-[1,3,5]triazine-2,4,6-triamineToxic by eye at 50and 10 uM noblunt/round justdeath 112

N2-Butyl-N4-(3-chloro-4-methoxy-phenyl)-N6-methyl-N6-piperidin-4-yl-N2-propyl-1,3,5-triazine-2,4,6-triamineToxic by eye at 50and 10 uM noblunt/round justdeath 136

N-(3-Chloro-4-methoxy-phenyl)-N′-cycloheptyl-N″-(1-ethyl-pyrrolidin-2-ylmethyl)-[1,3,5]triazine-2,4,6-triamineNothing but celldebris at 3 uMvery toxic 119

N2-(3-chloro-4-methoxyphenyl)-6-cyclohexylmethoxy-N4-methyl-N4-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4-diaminetoxic

TABLE 4B Triazine compounds toxic to endothelial cells when AGEstimulated % IL6 production % IL6 production compare to AGE compare toTNF stimulation of IL6 stimulation of IL6 (100%). (100%). CompdsCompound Name Generated Toxic Toxic Compds tested @ 10 uM tested @ 10 uMNumber STRUCTURE by AutonomTM (AGE-treated) (TNF-treated) HAEC, IL6HAEC, IL6 121

N,N′-Bis-(3-chloro-4-methoxy-phenyl)-N″-methyl-N″-(4-methyl-cyclohexyl)-[1,3,5]triazine-2,4,6-triaminetoxic n.d. total cell death @ 10 uM[50 @ 5 uM, but sometoxicity (~20%)]n.d. 168

N2-cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-N6-methyl-N6-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triaminecitratesalt toxic n.d. total cell death @ 10 uM[40 @ 5 uM] n.d. 159

N2-Cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-N6-methyl-N6-piperidin-4-yl-1,3,5-triazine-2,4,6-triaminetoxic n.d. total cell death @ 5 and10 uM n.d. 167

N2-cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-N6-methyl-N6-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triaminemaleatesalt toxic n.d. total cell death @ 10 uM[25 @ 5 uM] n.d. 169

N2-cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-N6-methyl-N6-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triaminesuccinatesalt toxic n.d. total cell death @ 10 uM[45 @ 5 uM n.d. 148

N-(1-Aza-bicyclo[2.2.2]oct-3-yl)-N′-(3-chloro-4-methoxy-phenyl)-N″-)1-ethyl-pyrrolidin-2-ylmethyl)-[1,3,5]triazine-2,4,6-triaminetoxic n.d. total cell death @ 10 uM[60 @ 5 uM] n.d. 134

N-(1-benzyl-piperidin-4-yl)-N′-(3-chloro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]-2,4,6-triaminetoxic n.d. total cell death @ 10 uM[55 @ 5 uM] n.d. 135

N2-(3-chloro-4-methoxy-phenyl)-N4-cycloheptyl-N6-piperidin-4-yl-1,3,5-triazine-2,4,6-triaminetoxic n.d. total cell death @ 10 uM[60 @ 5 uM] n.d. 138

2-chloro-4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl-amino]-1,3,5-triazin-2-ylamino}-phenoltoxic n.d. toxic n.d.

TABLE 5 Triazine compounds useful in treating inflammatory disordersgenerally, and all diseases related to inhibition of IL6. % IL6production % IL6 production compare to TNF compare to AGE Inhibition ofTNF- stimulation of IL6 stimulation of IL6 induced IL6 Inhibition ofTNF- (100%). Compds (100%). Compds Name production (See induced IL6tested @ 10 uM tested @ 10 uM Patent Number STRUCTURE Generated byAutonom (TM) NOTE) production HAEC, IL6 HAEC, IL6 76

N2-cycloheptyl-N4-ethyl-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ n.d. 18 n.d. 77

N2-(tert-butyl)-N4-cycloheptyl-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ n.d. 21 n.d. 78

N2-benzyl-N4-cycloheptyl-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine+++ n.d. 47 n.d. 82

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-6-(1-pyrrolidinyl)-1,3,5-triazine-2,4-diamine+++ n.d. 34 n.d. 83

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-6-hexahydro-1H-azepin-1-yl-1,3,5-triazine-2,4-diamine++++ n.d. 18 n.d. 84

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-6-octahydro-1(2H)-quinolinyl-1,3,5-triazine-2,4-diamine+++ n.d. 33 n.d. 85

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-N6-(4-methylcyclohexyl)-1,3,5-triazine-2,4,6-triamine+++ n.d. n.d. 86

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-6-[(2S)-2-(methoxymethyl)-1-pyrrolidinyl]-1,3,5-triazine-2,4-diamine++++ n.d. 19 n.d. 87

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-6-(4-methyl-1-piperazinyl)-1,3,5-triazine-2,4-diaminen.d. ++++ n.d. 5 88

6-(4-acetyl-1-piperazinyl)-N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4-diamine++++ n.d. 19 n.d. 91

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-N6-(2-furanylmethyl)-1,3,5-triazine-2,4,6-triamine+++ n.d. 28 n.d. 93

N2-cycloheptyl-N4-[2-(dimethylamino)ethyl]-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ ++++ 13 6 (IC50 = 3.36) 94

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-6-{4-[2-oxo-(1-pyrrolidinyl)ethyl]-1-piperazinyl]-1,3,5-triazine-2,4-diamine+++ ++++ 32 10 (IC50 = 5.8) 95

N-Cycloheptyl-N′-(1-ethyl-pyrrolidin-2-ylmethyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine++++ ++++ 5 (IC50 = 2.8) 1 (IC50 = 6.6) 96

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-N6-(2-(1-piperidinyl)ethyl]-1,3,5-triazine-2,4,6-triamine++++ n.d. 6 n.d. 99

1-[3-({4-(cycloheptylamino)-6-[(3-fluoro-4-methoxyphenyl)amino]-1,3,5-triazin-2-yl}amino)propyl]-2-pyrrolidinone+++ ++++ 39 8 (IC50 = 6.04) 100

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-N6-[3-(1H-imidazol-1-yl)propyl]-1,3,5-triazine-2,4,6-triaminen.d. ++++ n.d. 1 3

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(3-quinolinyl)-1,3,5-triazine-2,4,6-triaminen.d. ++++ n.d. 1 4

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(6-quinolinyl)-1,3,5-triazine-2,4,6-triaminen.d. ++++ n.d. 1 10

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(4-fluorophenyl)-1,3,5-triazine-2,4,6-triaminen.d. ++++ n.d. 1 11

4-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrroldinyl)methyl]amino}-1,3,5-triazin-2-yl)-amino]benzonitrilen.d. ++++ n.d. 1 (IC50 = 8.87) 24

N2-cycloheptyl-N4-(2,3-dihydro-1,4-benzodioxin-6-yl)-N6-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triaminen.d. ++++ n.d. 1 25

N2-cycloheptyl-N4-[4-(dimethylamino)phenyl]-N6-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triaminen.d. ++++ n.d. 1 26

N²-(3-chloro-4-diethylamino-phenyl)-N⁴-cycloheptyl-N⁶-(1-ethly-pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine++++ n.d. 1 (over 50% of cells werefloating n.d. 27

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-[4-(4-morpholinyl)phenyl]-1,3,5-triazine-2,4,6-triamine++++ ++++ 3 1 28

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-[4-(4-methyl-1-piperazinyl)phenyl]-1,3,5-triazine-2,4,6-triamine++++ n.d. 23 n.d. 29

N-{4-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-amino]phenyl}acetamide++++ ++++ 13 7 30

N-{3-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-amino]phenyl}acetamide++++ ++++ 9 17 32

N2-cycloheptyl-N4-(4-ethoxyphenyl)-N6-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triaminen.d. ++++ n.d. 1 34

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(2-pyridinyl)-1,3,5-triazine-2,4,6-triamine++++ ++++ 3 1 35

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(2-methylphenyl)-1,3,5-triazine-2,4,6-triamine+++ ++++ 34 4 39

2-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-amino]-4-methyl-3-thiophenecarboxamide n.d. +++ n.d. 28 40

N2-(4-chlorophenyl)-N4-cycloheptyl-N6-[(1-ethyl-2-pyrrolidinyl)methyl]-N2-methyl-1,3,5-triazine-2,4,6-triaminen.d. ++++ n.d. 19 41

3-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-(phenyl)amino]propanenitrilen.d. ++++ n.d. 18 42

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(4-methoxyphenyl)-N6-methyl-1,3,5-triazine-2,4,6-triamine++++ ++++ 1 1 43

N2-cycloheptyl-N4-(2,4-difluorophenyl)-N6-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-methyl-1,3,5-triazine-2,4,6-triaminen.d. ++++ n.d. 4 44

[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)(phenyl)amino]acetonitrilen.d. ++++ n.d. 1 (IC50 = 7.1) 45

N2-(3-chlorophenyl)-N4-cycloheptyl-N6-[(1-ethyl-2-pyrrolidinyl)methyl]-N2-methyl-1,3,5-triazine-2,4,6-triaminen.d. ++++ n.d. 1 46

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-methyl-N6-[2-(trifluoromethyl)phenyl]-1,3,5-triazine-2,4,6-triaminen.d. ++++ n.d. 3 48

N2-(3-chloro-4-methoxyphenyl)-N4-cycloheptyl-N6-[(1-ethyl-2-pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine+++ n.d. 3074 (IC50 = 4.3) n.d. 51

N2-ethyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ n.d. 1 n.d. 52

N2-(tert-butyl)-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ +++ 1 35 53

N2-benzyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ +++ 1 32 55

N2-cyclohexyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ n.d. 1 n.d. 56

N2-cyclopentyl-N4-[(1-ethyl-2-pyrrolidinyl)methy]-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ n.d. 1 n.d. 57

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-6-(1-pyrrolidinyl)-1,3,5-triazine-2,4-diamine++++ +++ 1 25 58

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-6-hexahydro-1H-azepin-1-yl-1,3,5-triazine-2,4-diamine++++ +++ 1 30 60

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-N6-(4-methylcyclohexyl)-1,3,5-triazine-2,4,6-triamine++++ n.d. 1 n.d. 61

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(4-fluoro-3-methoxyphenyl)-6-[2-(methoxymethyl)-1-pyrrolidinyl]-1,3,5-triazine-2,4-diamine++++ +++ 2 38 (IC50 = 6.47) 62

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-6-(4-methyl-1-piperazinyl)-1,3,5-triazine-2,4-diaminen.d. +++ n.d. 42 63

6-(4-acetyl-1-piperazinyl)-N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4-diamine+++ n.d. 26 n.d. 64

4-{4-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-6[(3-fluoro-4-methoxyphenyl)amino]-1,3,5-triazin-2-yl}-1-piperazinecarboxylate+++ n.d. 23 n.d. 65

N2-(cyclohexylmethyl)-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ +++ 1 (IC50 = 6.7) 45 (IC50 = 3.24) 66

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-N6-(2-furylmethyl)-1,3,5-triazine-2,4,6-triamine++++ ++++ 1 23 67

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-N6-(2,2,2-trifluoroethyl)-1,3,5-triazine-2,4,6-triaminen.d. +++ n.d. 39 68

N2-[2-(dimethylamino)ethyl]-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ +++ 1 40 (IC50 = 7.7) 69

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-6-{4-[2-oxo-2-(1-pyrrolidinyl)ethyl[-1-piperazinyl}-1,3,5-triazine-2,4-diamine++++ ++++ 1 5 70

N2,N4-bis[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,5-triamine++++ +++ 1 35 71

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-N6-[2-(1-piperidinyl)ethyl]-1,3,5-triazine-2,4,6-triamine++++ +++ 1 29 72

6-[4-(1,3-benzodioxol-5-ylmethyl)-1-piperazinyl]-N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4-diamine++++ +++ 5 31 73

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-6-[4-(2-pyridinyl)-1-piperazinyl]-1,3,5-triazine-2,4-diamine++++ ++++ 1 (IC50 = 8.7) 20 (IC50 = 7.5) 74

1-[3-({4-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-6-[(3-fluoro-4-methoxyphenyl)amino]-1,3,5-triazin-2-yl}amino)propyl]-2-pyrrolidinonen.d. +++ n.d. 49 75

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-N6-[3-(1H-imidazol-1-yl)propyl]-1,3,5-triazine-2,4,6-triamine+++ +++ 43 46 140

N²-cycloheptyl-N⁴-((R)-1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ n.d. 12.57 n.d. 139

N²-cycloheptyl-N⁴-((S)-1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ n.d. 16.08 n.d. 127

6-Chloro-N-cycloheheptyl-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine+++ n.d. 48 n.d. 141

N²-cyclohexylmethyl-N⁴-((S)-1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ n.d. 23.44 n.d. 142

N²-cyclohexylmethyl-N⁴-((R)-1-ethyl-pyrrolidin-2-ylmethyl)-N⁶-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++++ n.d. 21.62 n.d. 143

({4-cycloheptylamino-6-[((S)-1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile++++ n.d. 18.88 n.d. 144

({4-cycloheptylamino-6-[((R)-1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile++++ n.d. 157

N,N′-dicyclopropyl-N″-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine++ n.d. 60.48 n.d. 160

N²-cycloheptyl-N⁴-(3-fluoro-4-methoxyphenyl)-N⁶-methyl-N⁶-(1-methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine,hydrogenchloride salt ++++ n.d. 1 n.d. 101

(3-Chloro-4-methoxy-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-aminen.d. n.d. n.d. n.d. 107

N²-(3-chloro-4-methoxy-phenyl)-N⁴-isopropyl-N⁶-methyl-N⁶-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine++ n.d. 60 @ 5 uM n.d. 108

5-{4-(3-Chloro-4-methoxy-phenylamino)-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-[1,3,5]triazin-2-ylamino}-pentan-1-ol+++ n.d. 25 n.d. 109

5-[4-(3-chloro-4-methoxy-phenylamino)-6-(methyl-piperidin-4-yl-amino)-1,3,5-triazin-2-ylamino]-pentan-1-ol+++ n.d. 40 n.d. 111

N-Butyl-N′-(3-chloro-4-methoxy-phenyl)-N′-(1-methyl-piperidin-4-yl)-N-propyl-[1,3,5]triazine-2,4,6-triamine +n.d. >90 [46 @ 5 uM] did notsee toxicity n.d. 112

N²-Butyl-N⁴-(3-chloro-4-methoxy-phenyl)-N⁶-methyl-N⁶-piperidin-4-yl-N²-propyl-1,3,5-triazine-2,4,6-triamine +n.d. 90 @ 5 uM did not seetoxicity n.d. NOTE: The scale is as follows:(1) “++++” = 0-25% of IL6 production compared to cells that did notreceive compound (or per cent of control IL6 production); (2) “+++”= >25-50% of control IL6 production; and (3) “++” = >50-75% of controlIL6 production; and, (4) “+” = >75-100% of control IL6 production.“n.d.” means that the activity of the compound was not determined in thegiven assay.

TABLE 6 Inhibition of TNF-induced heparanase secretion Name RelativePatent # STRUCTURE Generated by Autonom ™ Activity Activity 76

N2-cycloheptyl-N4-ethyl-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine++ 50% inhibition @ 5 uM 82

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-6-(1-pyrrolidinyl)-1,3,5-triazine-2,4-diamine+++ 73% inhibition @ 5 uM 87

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-6-(4-methyl-1-piperazinyl)-1,3,5-triazine-2,4-diamine +40-60% inhibition @ 10 uM 94

N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-6-{4-[2-oxo-(1-pyrrolidinyl)ethyl]-1-piperazinyl}-1,3,5-triazine-2,4-diamine +40-60% inhibition @ 10 uM 35

N2-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(2-methylphenyl)-1,3,5-triazine-2,4,6-triamine++ 53% inhibition @ 5 uM 52

N2-(tert-butyl)-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine +40-60% inhibition @ 10 uM 58

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-6-hexahydro-1H-azepin-1-yl-1,3,5-triazine-2,4-diamine +40-60% inhibition @ 10 uM 61

N2-[(1-ethyl-2-pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-6-[2-(methoxymethyl)-1-pyrrolidinyl]-1,3,5-triazine-2,4-diamine +40-60% inhibition @ 10 uM NOTE: The scale is as follows: (1) “+++” =70-100% inhibition; (2) “++” = 30-40% inhibition; and (3) “+” = 0-30%inhibition all at 5 uM compound concentration.

TABLE 7 Triazine compounds useful in treating inflammatory disordersgenerally, and all diseases related to inhibition of IL6. % IL6production % IL6 production compare to TNF compare to AGE stimulation ofIL6 stimulation of IL6 Inhibition of TNF- Inhibition of TNF- (100%).Compds (100%). Compds Compound Name induced IL6 induced IL6 tested @ 10uM tested @ 10 uM Number STRUCTURE (IUPAC-Generated by Autonom)production production HAEC, IL6 HAEC, IL6 172

N-Cycloheptyl-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-naphthalen-2-yl-[1,3,5]triazine-2,4,6-triamine+++ ++ 18 n.d. 171

N-Cycloheptyl-N′-(3-fiuoro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine+++ ++ 21 n.d. 173

[4-(4-Benzyl-piperazin-1-yl)-6-morpholin-4-yl[1,3,5]triazin-2-yl]-(4-methoxy-phenyl)-amine++ 47 n.d. 174

N-Cycloheptyl-6-morpholin-4-yl-N′-naphthalen-2-yl-[1,3,5]triazine-2,4-diamine+++ 34 n.d. 175

N-Cycloheptyl-N′-(3-fluoro-4-methoxy-phenyl)-6-morpholin-4-yl-[1,3,5]triazine-2,4-diamine+++ 18 n.d. 176

N-Cycloheptyl-6-morpholin-4-yl-N′-phenyl-[1,3,5]triazine-2,4-diamine +++33 n.d. 177

N-Cycloheptyl-N-(4-methoxy-phenyl)-6-morpholin-4-yl-[1,3,5]triazine-2,4-diamine+++ ++ n.d. 178

N-Benzyl-N′-cycloheptyl-N″-(4-methoxy-phenyl)-N-methyl-[1,3,5]triazine-2,4,6-triamine++ 19 n.d. 179

N-(2-[1,3]Dioxolan-2-yl-ethyl)-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N″-naphthalen-2-yl-[1,3,5]triazine-2,4,6-triamine++ + 180

N-Cyclopropyl-N′-methyl-N′-(1-methyl-piperidin-4-yl)-N′-naphthalen-2-yl-[1,3,5]triazine-2,4,6-triamine+++ + n.d. 5 NOTE: “+++” represents between about 85 to 100% inhibitionof IL6 production in the presence of AGE or TNF, as compared to cellsthat did not receive any compound; “++” represents between about 65 andabout 85% inhibition of IL6 production in the presence of AGE or TNF;and “+” represents between about 50 and about 65% inhibition of IL6production in the presence of AGE or TNF.

TABLE 8 Bioactivity Data for Representative Compounds StructureProliferation Data Inflammation Data

Did not pass 5 & 10 uM screens;rounding Not Active

Did not pass 5 & 10 uM screens;blunting and rounding total cell death at10 uM(75% inhibition); 25% cell death at5 uM (not active)

Not active at 5 & 10 uM 48% inhibition at 10 uM with 20%cell death

Not active at 5 & 10 uM complete inhibition at 10 uM

Not active <25% inhibition at 10 uM

Not active at 5 & 10 uM 75% inhibition at 10 uM

4.9 uM average IC50 60% inhibition at 10 uM with 20%cell death

1.85 uM average IC50 85% inhibition at 10 uM with 20%cell death

5.25 uM average IC50 total cell death at 10 uM (completeinhibition)

rounding at 5 & 10 uM total cell death at 10 uM(95% inhibition)

Not active Not Active

Not active Not Active

Not active Not Active

Not active 60% inhibition at 10 uM

6.37 uM; cell morphology changeat 5 & 10 uM; no cell death Not active at1 or 5 uM; 30% celldeath at 5 uM and total death at 10uM

6.1 uM: cell morphology changeat 7.5 uM and cell death at 10 uM totalcell death at 10 uM; 58%inhibition at 5 uM, but may be dueto celldeath(50% death), notactive at 1 uM

5.5 uM total cell death at 10 uM; 50%inhibition at 5 uM with 20%celldeath

Not active 35% inhibition at 10 uM

Not active 25% inhibition at 5 uM and 10 uM

3.3 uM; 4.5 uM; cell blunting at 3to 10 uM total cell death at 10 uM(84%inhibition); 64% inhibition at 5 uM

1.5 uM; 4.2 uM; cell blunting at 3uM and rounding at 5 uM total celldeath at 10 uM (97%inhibition ); 43% inhibition at 5 uMwith 25% celldeath

1.9 uM; cell blunting at 1 uM androunding at 5 uM 41% inhibition at 10uM with >50%cell death; 37% inhibition at 5 uMwith 20% cell death

6.6 uM 88% inhibition at 10 uM with >50%cell death; 69% inhibition at 5uMwith 20% cell death

Not active 36% inhibition at 10 uM

Not active 25% inhibition at 10 uM

Not active 23% inhibition at 10 uM

Not tested Not tested

4.1 uM Not active with 15% cell death at10 uM

Not active 20% inhibition at 10 uM, no effectat 5 uM

7.8 uM; 5.3 uM 35% inhibition at 10 uM, 25%inhibition at 5 uM

1. A compound having the formula:

or a salt, a diastereomeric mixture, an enantiomer, a tautomer, or aracemic mixture thereof; wherein: G is NH or NMe; E is N or CH; X¹ isselected from m-F, m-Cl, m-Br, m-l, m-NO₂, m-CN, m-CF₃, m-C(O)OEt, orm-C(O)ONa; X² is selected from p-OCH₃, p-OEt, p-O-n-Pr, p-O-i-Pr,p-OCF₃, p-OC(O)Me, p-NH₂, p-NHMe, p-NMe₂, p-Me, p-Et, p-OH, p-ONa,p-OP(O)(OH)₂, p-OCH₂OP(O)(OH)₂, p-OCH₂OP(O)(OH)(ONa),p-OCH₂OP(O)(O-t-Bu)₂, p-OCH₂OP(O)(OCH₂Ph)₂, p-OCH₂SMe, p-OCH₂OMe,p-O(CH₂CH₂O)₃CH₂CH₂OH, p-OC(O)(CH₂CH₂O)₄CH₂CH₂OH, orp-OC(O)(CH₂CH₂O)₄CH₂CH₂OMe, R¹ is H; z is 0, 1, or 2; either: a) A is NHor NMe, and Y¹ is selected from

or b) AY¹ in combination is selected from

either: a) D is NH, NMe, or O, and Y² is selected from —H,

or b) DY² in combination is selected from


2. A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and one or more compounds according to claim
 1. 3. Thepharmaceutical composition as claimed in claim 2, further comprising: apharmaceutically acceptable auxiliary; a pharmaceutically acceptablepreservative; a pharmaceutically acceptable excipient; or anycombination thereof.
 4. The pharmaceutical composition as claimed inclaim 2, wherein the pharmaceutical composition is in the form of atablet, a capsule, a cachet, a powder, a granule, a solution, asuspension, an emulsion, a bolus, a lozenge, a suppository, a pessary, atampon, a cream, a gel, a paste, a foam, a spray, an aerosol, amicrocapsule, a liposome, a transdermal patch, a pastille, a paste, or amouthwash.
 5. A compound having the formula:

or a salt, a diastereomeric mixture, an enantiomer, a tautomer, or aracemic mixture thereof; wherein: R^(1a) is selected from

R^(6a) is selected from H or Me; R^(x) is selected from

wherein R^(2a) is selected from H or Me; and R^(3a) is selected from H,


6. A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and one or more compounds according to claim
 5. 7. Thepharmaceutical composition as claimed in claim 6, further comprising: apharmaceutically acceptable auxiliary; a pharmaceutically acceptablepreservative; a pharmaceutically acceptable excipient; or anycombination thereof.
 8. The pharmaceutical composition as claimed inclaim 6, wherein the pharmaceutical composition is in the form of atablet, a capsule, a cachet, a powder, a granule, a solution, asuspension, an emulsion, a bolus, a lozenge, a suppository, a pessary, atampon, a cream, a gel, a paste, a foam, a spray, an aerosol, amicrocapsule, a liposome, a transdermal patch, a pastille, a paste, or amouthwash.
 9. A compound having the formula:

or a salt, a diastereomeric mixture, an enantiomer, a tautomer, or aracemic mixture thereof; wherein: R^(1a) is selected from

R^(7a) is selected from

wherein R^(4a) is selected from H or Me; and R^(5a) is selected from


10. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and one or more compounds according to claim
 9. 11.The pharmaceutical composition as claimed in claim 10, furthercomprising: a pharmaceutically acceptable auxiliary; a pharmaceuticallyacceptable preservative; a pharmaceutically acceptable excipient; or anycombination thereof.
 12. The pharmaceutical composition as claimed inclaim 10, wherein the pharmaceutical composition is in the form of atablet, a capsule, a cachet, a powder, a granule, a solution, asuspension, an emulsion, a bolus, a lozenge, a suppository, a pessary, atampon, a cream, a gel, a paste, a foam, a spray, an aerosol, amicrocapsule, a liposome, a transdermal patch, a pastille, a paste, or amouthwash.
 13. A compound having the formula:

or a salt, a diastereomeric mixture, an enantiomer, a tautomer, or aracemic mixture thereof; wherein: R^(1a) is selected from

R^(2a) is selected from

R^(3a) is selected from —H,

R^(5a) is selected from

R^(6a) is selected from —H or —CH₃.
 14. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and one or morecompounds according to claim
 13. 15. The pharmaceutical composition asclaimed in claim 14, further comprising: a pharmaceutically acceptableauxiliary; a pharmaceutically acceptable preservative; apharmaceutically acceptable excipient; or any combination thereof. 16.The pharmaceutical composition as claimed in claim 14, wherein thepharmaceutical composition is in the form of a tablet, a capsule, acachet, a powder, a granule, a solution, a suspension, an emulsion, abolus, a lozenge, a suppository, a pessary, a tampon, a cream, a gel, apaste, a foam, a spray, an aerosol, a microcapsule, a liposome, atransdermal patch, a pastille, a paste, or a mouthwash.
 17. A compoundselected from:N-(4-benzyloxy-3-chloro-phenyl)-N′-cycloheptyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine,N-(4-benzyloxy-3-chloro-phenyl)-N′-cycloheptyl-N″-methyl-N″-piperidin-4-yl-[1,3,5]triazine-2,4,6-triamine,4-[4-cycloheptylamino-6-(methyl-piperidin-4-yl-amino)-[1,3,5]triazin-2-ylamino]-phenol,4-{4-cycloheptylamino-6-[methyl-(1-methyl-piperidin-4-yl)-amino]-1,3,5]triazin-2-ylamino}-phenol,2-chloro-4-(4,6-dichloro-[1,3,5]triazin-2-ylamino)-phenol,2-chloro-4-(4-chloro-6-cycloheptylamino-[1,3,5]triazin-2-ylamino)-phenol,N-(1-benzyl-piperidin-4-yl)-N′-(3-fluoro-4-methoxy-phenyl)-N″-cycloheptyl-[1,3,5]triazine-2,4,6-diamine,N-cycloheptyl-N′-(4-methoxy-phenyl)-N″-piperidin-4-yl-[1,3,5]triazine-2,4,6-triamine,6-chloro-N-cyclopropyl-N′-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine,N-cyclopropyl-N′-(3-flouro-4-methoxy-phenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine,N-cyclopropyl-N′-(1-ethyl-pyrrolidin-2-ylmethyl)-N″-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine,6-chloro-N-(3-chloro-4-methoxyphenyl)-N′-cyclopropyl-[1,3,5]triazine-2,4-diamine,N-cyclopropyl-N′-(3-chloro-4-methoxyphenyl)-N″-methyl-N″-(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine,6-chloro-N,N′-bis-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-diamine,N-(1-ethyl-pyrrolidin-2-ylmethyl)-N′,N″-bis-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine,1-[4-(3-chloro-4-methoxyanilino)-cycloheptylamino-1,3,5-triazin-2-yl]-2-azoloamymethanol,N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(4-methylpiperzino)-1,3,5-triazine-4,2-diamine,3-[4-(3-chloro-4-methoxyanilino)-6-cycloheptylamino-1,3,5-triazin-2-yloxy]-2-ethyl-4H-4-pyranone,1-[3-{4-(3-chloro-4-methoxyanilino)-6-cycloheptylamino-1,3,5-triazine-2-yloxy}piperidino]-1-ethanone,N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-isopropoxy-1,3,5-triazine-2,4-diamine,N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(2-azolanylmethoxy)-1,3,5-triazine-2,4-diamine,1-[4-(3-chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazine-2-yl]-piperidin-3-ol],1-[4-(3-chloro-4-methoxy-phenylamino)-6-cycloheptylamino-[1,3,5]triazine-2-yl]-piperidin-4-ol],N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(1-methyl-2-azolanylmethoxy)-1,3,5-triazine-2,4-diamine,N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(1-methyl-4-piperidyloxy)-1,3,5-triazine-2,4-diamine,N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(1,4-thiazinan-4-yl)-1,3,5-triazine-4,2-diamine,N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(2-fluorophenoxy)-1,3,5-triazine-4,2-diamine,N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-[2-(2-fluorophenoxy)ethoxy]-1,3,5-triazine-4,2-diamine,N²-(3-chloro-4-methoxyphenyl)-N⁴-cycloheptyl-6-(6-methyl-2-pyrylmethoxy]-1,3,5-triazine-4,2-diamine,N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methylpiperidin-4-yl)[1,3,5]triazine-2,4,6-triamine,N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methylpiperidin-4-yl)[1,3,5]triazine-2,4,6-triamine, HCl salt,6-methoxy-N-(3-Chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4 diamine,6-ethoxy-N-(3-Chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4 diamine,6-isopropoxy-N-(3-Chloro-4-methoxyphenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4 diamine,N-(3-chloro-4-methoxyphenyl)-N′-cycloheptyl-N″-methyl-N″-(1-methylpiperidin-4-yl)[1,3,5]triazine-2,4,6-triamine,6-chloro-N-(3-chloro-4-methoxy-phenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine,(3-chloro-4-methoxy-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine,6-Chloro-N-(3-fluoro-4-methoxy-phenyl)-N′-cycloheptyl-[1,3,5]triazine-2,4-diamine,or (3-fluoro-4-methoxy-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine.18. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and according to claim
 17. 19. The pharmaceuticalcomposition as claimed in claim 18, further comprising: apharmaceutically acceptable auxiliary; a pharmaceutically acceptablepreservative; a pharmaceutically acceptable excipient; or anycombination thereof.
 20. The pharmaceutical composition as claimed inclaim 18, wherein the pharmaceutical composition is in the form of atablet, a capsule, a cachet, a powder, a granule, a solution, asuspension, an emulsion, a bolus, a lozenge, a suppository, a pessary, atampon, a cream, a gel, a paste, a foam, a spray, an aerosol, amicrocapsule, a liposome, a transdermal patch, a pastille, a paste, or amouthwash.
 21. A compound selected from:


22. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and one or more compounds according to claim
 21. 23.The pharmaceutical composition as claimed in claim 22, furthercomprising: a pharmaceutically acceptable auxiliary; a pharmaceuticallyacceptable preservative; a pharmaceutically acceptable excipient; or anycombination thereof.
 24. The pharmaceutical composition as claimed inclaim 22, wherein the pharmaceutical composition is in the form of atablet, a capsule, a cachet, a powder, a granule, a solution, asuspension, an emulsion, a bolus, a lozenge, a suppository, a pessary, atampon, a cream, a gel, a paste, a foam, a spray, an aerosol, amicrocapsule, a liposome, a transdermal patch, a pastille, a paste, or amouthwash.